Myosin light chain kinases: Division of work in cell migration

Cell motility is a highly coordinated multistep process. Uncovering the mechanism of myosin II (MYO2) activation responsible for the contractility underlying cell protrusion and retraction provides clues on how these complementary activities are coordinated. Several protein kinases have been shown to activate MYO2 by phosphorylating the associated myosin light chain (MLC). Recent work suggests that these MLC kinases are strategically localized to various cellular regions during cell migration in a polarized manner. This localization of the kinases together with their specificity in MLC phosphorylation, their distinct enzymatic properties and the distribution of the myosin isoforms generate the specific contractile activities that separately promote the cell protrusion or retraction essential for cell motility.Key words: myosin, MLCK, ROK, MRCK, phosphorylation, cell migrationCell movement is a fundamental activity underlying many important biological events ranging from embryological development to immunological responses in the adult. A typical cell movement cycle entails polarization, membrane protrusion, formation of new adhesions, cell body translocation and finally rear retraction.¹ A precise temporal and spatial coordination of these separate steps that take place in different parts of the cell is important for rapid and efficient movement.²One major event during eukaryotic cell migration is the myosin II (MYO2)-mediated contraction that underlies cell protrusion, traction and retraction.^1,3 An emerging theme from collective findings is that there are distinct myosin contractile modules responsible for the different functions which are separately regulated by local myosin regulatory light chain (MLC) kinases. These kinases contribute to contractile forces that connect adhesion, protrusion and actin organization.² Unraveling the regulation of these contractile modules is therefore pivotal to a better understanding of the coordination mechanism.At the lamellipodium, the conventional calcium/calmodulin-dependent myosin light chain kinase (MLCK) has been shown to play an essential role in a Rac-dependent lamellipodial extension.⁴ Inhibition of calmodulin or MLCK activity by specific photoactivatable peptides in motile eosinophils effectively blocks lamellipodia extension and net movement.⁵ Furthermore, there is a strong correlation between activated MLCK and phosphorylated MLC within the lamellipodia of Ptk-2 cells as revealed by fluorescence resonance energy transfer (FRET) analysis.⁶ More recent studies showed MLCK to regulate the formation of focal complexes during lamellipodia extension.^7,8 Functionally, MLCK is thought to play a critical role in the environment-sensing mechanism that serves to guide membrane protrusion. It mediates contraction that exerts tension on integrin-extracellular matrix (ECM) interaction, which, depending on the rigidity of the substratum, will lead to either stabilization of adhesion resulting in protrusion or destabilization of attachment seen as membrane ruffling on non-permissive surfaces.^8,9As a Rho effector, Rho-associated kinase (ROK/ROCK/Rho-kinase) has been shown to regulate stress fibers and focal adhesion formation by activating myosin, an effect that can be blocked by the specific ROK inhibitor Y-27632.^10,11 Myosin activation by ROK is the effect of two phosphorylation events: the direct phosphorylation on MLC and the inhibition of myosin phosphatase through phosphorylation of its associated myosin-binding subunit (MBS).¹¹ Consistent with this notion of a localization-function relationship, ROK and MBS, which can interact simultaneously with activated RhoA,¹¹ have been shown to colocalize on stress fibers.^12,13 In migrating cells, Rho and ROK activities have been mostly associated with the regulation of tail retraction, as inhibition of their activities often results in trailing tails due to the loss of contractility specifically confined to the cell rear.^14,15 Tail retraction requires high contractile forces to overcome the strong integrin-mediated adhesion established at the rear end, an event which coincides with the strategic accumulation of highly stable and contractile stress fibers that assemble at the posterior region of migrating cells.MRCK was previously shown to phosphorylate MLC and promote Cdc42-mediated cell protrusion.¹⁶ More recently, it was found to colocalize extensively with and regulate the dynamics of a specific actomyosin network located in the lamella and cell center, in a Cdc42-dependent manner but independent of MLCK and ROK.¹⁷ The lamellar actomyosin network physically overlaps with, but is biochemically distinct from the lamellipodial actin meshwork.^9,18 The former network consists of an array of filaments assembled in an arrangement parallel to the leading edge, undergoing continuous retrograde flow across the lamella, with their disassembly occurring at the border of the cell body zone sitting in a deeper region.^17–19 Retrograde flow of the lamellar network plays a significant role in cell migration as it is responsible for generating contractile forces that support sustained membrane protrusion and cell body advancement.^17–19It is therefore conceivable that these three known MLC kinases are regulated by different signaling mechanisms at different locations and on different actomyosin contractile modules. The coordination of the various modules will ensure persistent directional migration (Figure 1). Phosphorylation of MLC by PAK and ZIP kinase has also been reported, but their exact roles in this event have yet to be determined.^20,21 It is also noteworthy that individual kinases can work independently of each other, as amply shown by evidence from inhibitor treatments. This is particularly true for MRCK in the lamella, whose activity on lamellar actomyosin flow is not affected by ML7 and Y-27632, the inhibitors of MLCK and ROK respectively.¹⁷ These findings further indicate that although both ROK and MRCK have been shown to upregulate phosphorylated MLC levels by inhibiting the myosins phosphatases,^11,22 they are likely to act as genuine MLC kinases themselves, without the need of MLCK as previously suggested.¹¹Open in a separate windowFigure 1Upper panel depicts a model for the specific activation of the different MLC kinases at various locations in the cell. In response to upstream signals, MLC kinases MLCK, MRCK and ROK are activated and localized to different regions. In the case of MRCK and ROK, the interaction of the GTP-bound Rho GTPase binding domain will determine the specific action of the downstream kinase, resulting in actomyosin contractility at different locations. The coordination of these signalling events is crucial for directional cell migration. Lower panel shows a typical front-rear location for Myosin 2A and 2B in a migrating U2OS cell.In conjunction with their differences in localization, the three MLC kinases show apparent individual preferences and specificity towards the MYO2 isoforms that they associate with. The two major MYO2 isoforms MYO2A and 2B are known to have distinct intracellular distributions that are linked to their individual functions (Figure 1).^23,24 In motile cells, MYO2A localization that is skewed towards the protruding cell front is consistent with it being the major myosin 2 component of the lamellar filaments regulated by MRCK as well as its regulation by MLCK in lamellipodial contraction.^8,17,19 In contrast, the enrichment of MYO2B at retracting cell rear conforms well with the requirement of thick and stable stress fibers capable of causing tail contraction and prevention of protrusion under the control of Rho/ROK signaling.^23,25 The selection for MYO2B filaments in the cell rear stems from their more contractile and stable nature compared with MYO2A, a consequence of their higher time-averaged association with actin.^26,27 Conversely, the lower tension property of MYO2A filaments suggests that they are more dynamic in nature,^26,27 a characteristic which fits well with the dynamic actomyosin activities at the leading edge and lamella that regulate protrusion.It deserves special mention that the three MLC kinases display subtle differences in their specificity towards MLC. While MLCK and MRCK phosphorylate only a single Ser19 site (monophosphorylation),^18,28 ROK is able to act on both Thr18 and Ser19 residues causing diphosphorylation of MLC,²⁹ MLCK only causes diphosphorylation when present at higher concentrations.³⁰ By further increasing its actin-activated ATPase activity, diphosphorylation of MLC has been shown to induce a higher myosin activation and filament stability.^30–32 The use of specific antibodies that can differentiate between the two populations of phosphorylated MLC has been instrumental in revealing their localization and correlation with the activity of the MLC kinases. The emerging picture from these experiments is that mono and diphosphorylated MLC exhibit distinct distributions in migrating cells, with the monophosphorylated MLC localized more towards the protrusive region, while the diphosphorylated form is more enriched at the posterior end.^21,33 Taking into account their biochemical properties, the polarized distributions of these differentially phosphorylated MLC coincide functionally with the segregation of the MYO2 isoforms and their corresponding regulators. These findings provide further support for the existence of segregated contractile modules in migrating cell and their distinctive regulation.The mechanisms that determine the specific segregation of the contractile modules and their regulation are unclear. However, some clues have emerged from recent studies. It has been shown that the C-terminal coiled-coil region of MYO2B is important for determining its localization in cell rear²⁵ and which requires Rho/ROK activity as their inhibition resulted in the loss of this specific localization.²³ Correspondingly, the inhibition of MRCK activity resulted in the loss of lamella-localized MYO2A.¹⁷ These findings suggest that activation of MYO2 filaments by their upstream regulators is important for their functional segregation and maintenance. It is noteworthy that both ROK and MRCK have distinct regulatory domains including the pleckstrin homology domains which have been shown to be essential for their localization, a process which may involve myosin interaction and lipid-dependent targeting as has been respectively shown for ROK and MRCK.^11,13,16 Further, the specificity of MRCK for lamellar actomyosin is believed to be largely determined by the two proteins it forms a complex with: the adaptor LRAP35a, and the MYO2-related MYO18A. Activation of MYO18A by MRCK, a process bridged by LRAP35a, is a crucial step which facilitates MRCK regulation on lamellar MYO2A.¹⁷The mechanisms responsible for segregating the contractile modules and their regulators may also comprise a pathway that parallels the microtubule-modulatory Par6/aPKC/GSK3β signalling pathway which regulates cellular polarization. This notion is supported by both Cdc42 and Rho being common upstream regulators of these two pathways.³⁴ GTPase activation may determine the localized activities of the separate contractile modules and create an actomyosin-based asymmetry across the cell body, which together with the microtubule-based activities, result in the formation of a front-back axis important for directional movement. The involvement of MRCK in MTOC reorientation and nuclear translocation events,³⁵ and our unpublished observation that LRAP35a has a GSK3β-dependent microtubule stabilizing function are supportive of a possible cross-talk between these two pathways.In conclusion, the complex regulation of contractility in cell migration emphasizes the importance of the localization, specificity and enzymatic properties of the different MLC kinases and myosin isoforms involved. The initial excitement and confusion caused by the emergence of the different MLC kinases are fading, being now overtaken by the curiosity about how they cooperate and are coordinated while promoting cell motility.     

Prions: En route from structural models to structures

The prion hypothesis^1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.^4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.^6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.⁶ For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.Key words: prion, NMR, solid-state NMR, MAS, structure, Ure2p, HET-sDespite the large interest in the basic mechanisms of fibril formation and prion propagation, little is known about the molecular structure of prions at atomic resolution and the mechanism of propagation. Prions with related properties to the ones responsible for mammalian diseases were also discovered in yeast and funghi^8,9 which provide convenient model system for their studies. Prion proteins described include the mammalian prion protein PrP, Ure2p,¹⁰ Rnq1p,¹¹ Sup35,¹² Swi1,¹³ and Cyc8,¹⁴ from bakers yeast (S. cervisiae) and HET-s from the filamentous fungus P. anserina. The soluble non-prion form of the proteins characterized in vitro is a globular protein with an unfolded, dynamically disordered N- or C-terminal tail.^15–18 In the prion form, the proteins form fibrillar aggregates, in which the tail adopts a different conformation and is thought to be the dominant structural element for fibril formation.Fibrills are difficult to structurally characterize at atomic resolution, as X-ray diffraction and liquid-state NMR cannot be applied because of the non-crystallinity and the mass of the fibrils. Solid-state NMR, in contrast, is nowadays well suited for this purpose. The size of the monomer, between 230 and 685 amino-acid residues for the prions of Figure 1, and therefore the number of resonances in the spectrum—that used to be large for structure determination—is now becoming tractable by this method.Open in a separate windowFigure 1Prions identified today and characterized as consisting of a prion domain (blue) and a globular domain (red).Prion proteins characterized so far were found to be usually constituted of two domains, namely the prion domain and the globular domain (see Fig. 1). This architecture suggests a divide-and-conquer approach to structure determination, in which the globular and prion domain are investigated separately. In isolation, the latter, or fragments thereof, were found to form β-sheet rich structures (e.g., Ure2p(1-89),^6,19 Rnq1p(153-405)²⁰ and HET-s(218-289)²¹). The same conclusion was reached by investigating Sup35(1-254).²² All these fragements have been characterized as amyloids, which we define in the sense that a significant part of the protein is involved in a cross-beta motif.²³ An atomic resolution structure however is available presently only for the HET-s prion domain, and was obtained from solid-state NMR²⁴ (vide infra). It contains mainly β-sheets, which form a triangular hydrophobic core. While this cross-beta structure can be classified as an amyloid, its triangular shape does deviate significantly from amyloid-like structures of smaller peptides.²³Regarding the globular domains, structures have been determined by x-ray crystallography (Ure2p^25,26 and HET-s²⁷), as well as NMR (mammal prions^15,28–30). All reveal a protein fold rich in α-helices, and dimeric structures for the Ure2 and HET-s proteins. The Ure2p fold resembles that of the β-class glutathione S-transferases (GST), but lacks GST activity.²⁵It is a central question for the structural biology of prions if the divide-and-conquer approach imposed by limitations in current structural approaches is valid. Or in other words: can the assembly of full-length prions simply be derived from the sum of the two folds observed for the isolated domains?     

Structural basis of transmembrane domain interactions in integrin signaling

Cell surface receptors of the integrin family are pivotal to cell adhesion and migration. The activation state of heterodimeric αβ integrins is correlated to the association state of the single-pass α and β transmembrane domains. The association of integrin αIIbβ3 transmembrane domains, resulting in an inactive receptor, is characterized by the asymmetric arrangement of a straight (αIIb) and tilted (β3) helix relative to the membrane in congruence to the dissociated structures. This allows for a continuous association interface centered on helix-helix glycine-packing and an unusual αIIb(GFF) structural motif that packs the conserved Phe-Phe residues against the β3 transmembrane helix, enabling αIIb(D723)β3(R995) electrostatic interactions. The transmembrane complex is further stabilized by the inactive ectodomain, thereby coupling its association state to the ectodomain conformation. In combination with recently determined structures of an inactive integrin ectodomain and an activating talin/β complex that overlap with the αβ transmembrane complex, a comprehensive picture of integrin bi-directional transmembrane signaling has emerged.Key words: cell adhesion, membrane protein, integrin, platelet, transmembrane complex, transmembrane signalingThe communication of biological signals across the plasma membrane is fundamental to cellular function. The ubiquitous family of integrin adhesion receptors exhibits the unusual ability to convey signals bi-directionally (outside-in and inside-out signaling), thereby controlling cell adhesion, migration and differentiation.^1–5 Integrins are Type I heterodimeric receptors that consist of large extracellular domains (>700 residues), single-pass transmembrane (TM) domains, and mostly short cytosolic tails (<70 residues). The activation state of heterodimeric integrins is correlated to the association state of the TM domains of their α and β subunits.^6–10 TM dissociation initiated from the outside results in the transmittal of a signal into the cell, whereas dissociation originating on the inside results in activation of the integrin to bind ligands such as extracellular matrix proteins. The elucidation of the role of the TM domains in integrin-mediated adhesion and signaling has been the subject of extensive research efforts, perhaps commencing with the demonstration that the highly conserved GFFKR sequence motif of α subunits (Fig. 1), which closely follows the first charged residue on the intracellular face, αIIb(K989), constrains the receptor to a default low affinity state.¹¹ Despite these efforts, an understanding of this sequence motif had not been reached until such time as the structure of the αIIb TM segment was determined.¹² In combination with the structure of the β3 TM segment¹³ and available mutagenesis data,^6,9,10,14,15 this has allowed the first correct prediction of the overall association of an integrin αβ TM complex.¹² The predicted association was subsequently confirmed by the αIIbβ3 complex structure determined in phospholipid bicelles,¹⁶ as well as by the report of a similar structure based on molecular modeling using disulfide-based structural constraints.¹⁷ In addition to the structures of the dissociated and associated αβ TM domains, their membrane embedding was defined^{12,13,16,18,19} and it was experimentally recognized that, in the context of the native receptor, the TM complex is stabilized by the inactive, resting ectodomain.¹⁶ These advances in integrin membrane structural biology are complemented by the recent structures of a resting integrin ectodomain and an activating talin/β cytosolic tail complex that overlap with the αβ TM complex,^20,21 allowing detailed insight into integrin bi-directional TM signaling.Open in a separate windowFigure 1Amino acid sequence of integrin αIIb and β3 transmembrane segments and flanking regions. Membrane-embedded residues^{12,13,16,18,19} are enclosed by a gray box. Residues 991–995 constitute the highly conserved GFFKR sequence motif of integrin α subunits.     

Leaf carbohydrate metabolism during defense: Intracellular sucrose-cleaving enzymes do not compensate repression of cell wall invertase

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild type (wt) tobacco Nicotiana tabacum L. Samsun NN (SNN) with plants with RNA interference-mediated repression of cwINV (SNN::cwINV) during the interaction with the oomycetic phytopathogen Phytophthora nicotianae. We have previously shown that the transgenic plants developed normally under standard growth conditions, but exhibited weaker defense reactions in infected source leaves and were less tolerant to the pathogen. Here, we show that repression of cwINV was not accompanied by any compensatory activities of intracellular sucrose-cleaving enzymes such as vacuolar and alkaline/neutral invertases or sucrose synthase (SUSY), neither in uninfected controls nor during infection. In wt source leaves vacuolar invertase did not respond to infection, and the activity of alkaline/neutral invertases increased only slightly. SUSY however, was distinctly stimulated, in parallel to enhanced cwINV. In SNN::cwINV SUSY-activation was largely repressed upon infection. SUSY may serve to allocate sucrose into callose deposition and other carbohydrate-consuming defense reactions. Its activity, however, seems to be directly affected by cwINV and the related reflux of carbohydrates from the apoplast into the mesophyll cells.Key words: cell wall invertase, apoplastic invertase, alkaline invertase, neutral invertase, sucrose synthase, plant defense, Nicotiana tabacum, Phytophthora nicotianaePlant defense against pathogens is costly in terms of energy and carbohydrates.^1,2 Sucrose (Suc) and its cleavage products glucose and fructose are central molecules for metabolism and sensing in higher plants (reviewed in refs. ³ and ⁴). Rapid mobilization of these carbohydrates seems to be an important factor determining the outcome of plant-pathogen interactions. In particular in source cells reprogramming of the carbon flow from Suc to hexoses may be a crucial process during defense.^1,2There are two alternative routes of sucrolytic carbohydrate mobilization. One route is reversible and involves an uridine 5′-diphosphate (UDP)-dependent cleavage catalyzed by sucrose synthase (SUSY). Its activity is limited by the concentrations of Suc and UDP in the cytosol, as the affinity of the enzyme to its substrate is relatively low (K_m for Suc 40–200 mM). The other route is the irreversible, hydrolytic cleavage by invertases (INVs), which exhibit high affinity to Suc (K_m 7–15 mM).⁵Plants possess three different types of INV isoenzymes, which can be distinguished by their solubility, subcellular localization, pH-optima and isoelectric point. Usually, they are subdivided into cell wall (cwINV), vacuolar (vacINV), and alkaline/neutral (a/nINVs) INVs.cwINV, also referred to as extracellular or apoplastic INV, is characterized by a low pH-optimum (pH 3.5–5.0) and usually ionically bound to the cell wall. It is the key enzyme of the apoplastic phloem unloading pathway and plays a crucial role in the regulation of source/sink relations (reviewed in refs. ^{3, 6–8}). A specific role during plant defense has been suggested, based on observations that cwINV is often induced during various plant-pathogen interactions, and the finding that overexpression of a yeast INV in the apoplast increases plant resistance.^6,8–10 It was shown, that a rapid induction of cwINV is, indeed, one of the early defense-related reactions in resistant tobacco source leaves after infection with Phytophthora nicotianae (P. nicotianae).¹¹ Finally, the whole infection area in wt leaves was covered with hypersensitive lesions, indicating that all cells had undergone hypersensitive cell death (Fig. 1A).^1,11 When the activity of cwINV was repressed by an RNAi construct, defense-related processes were impaired, and the infection site exhibited only small spots of hypersensitive lesions. Finally, the pathogen was able to sporulate, indicating a reduced resistance of these transgenic plants (Fig. 1A).¹Open in a separate windowFigure 1Defense-induced changes in the activity of intracellular sucrose-cleaving enzymes and their contribution to defense. (A) The repression of cwINV in source leaves of tobacco leads to impaired pathogen resistance and can not be compensated by other sucrose-cleaving enzymes. The intensity of defense reactions is amongst others indicated by the extent of hypersensitive lesions. (B and C) Absolute activity of vacuolar (B) and alkaline/neutral (C) INVs at the infection site (white symbols, control; black symbols, infection site). (D) Increase in SUSY activity at the infection site. All data points taken from noninfected control parts of the plants in each individual experiment and each point along the time scale of an experiment are set as 0%. At least three independent infections are averaged and their means are presented as percentage changes ± SE (circles, SNN; triangles, SNN::cwINV). Insets show the means of the absolute amount of activities (white symbols, control; black symbols, infection site). Material and methods according to Essmann, et al.¹vacINV, also labeled as soluble acidic INV, is characterized by a pH optimum between pH 5.0–5.5. Among others it determines the level of Suc stored in the vacuole and generates hexose-based sugar signals (reviewed in refs. ³ and ¹²). Yet, no specific role of vacINV during pathogen response has been reported. Although vacINV and cwINV are glycoproteins with similar enzymatic and biochemical properties and share a high degree of overall sequence homology and two conserved amino acid motifs,⁴ the activity of vacINV in tobacco source leaves was not changed due to the repression of the cwINV (Fig. 1B).¹ After infection with P. nicotianae the activity of vacINV in wt SNN did not respond under conditions where cwINV was stimulated.¹ There was also no significant change in the transgenic SNN::cwINV (Fig. 1B). This suggests that during biotic stress, there is no crosstalk between the regulation of cwINV and vacINV.a/nINVs exhibit activity maxima between pH 6.5 and 8.0, are not glycosylated and thought to be exclusively localized in the cytosol. But recent reports also point to a subcellular location in mitochondria and chloroplasts.^13,14 Only a few a/nINVs have been cloned and characterized, and not much is known about their physiological functions (reviewed in refs. ^{4, 14} and ¹⁵). Among other things they seem to be involved in osmotic or low-temperature stress response.^14,15 During the interaction between tobacco and P. nicotianae the activity of a/nINVs rose on average 17% in the resistant wt SNN between 1 to 9 hours post infection (Fig. 1C). By contrast, in SNN::cwINV the a/nINVs activities remained unchanged in control leaves and even after infection (Fig. 1C). This suggests that the defense related stimulation in a/nINVs activities is rather a secondary phenomenon, possibly in response to the enhanced cwINV activity and the related carbohydrate availability in the cytosol.SUSY can be found as a soluble enzyme in the cytosol, bound to the inner side of the plasma membrane or the outer membrane of mitochondria, depending on the phosphorylation status. It channels hexoses into polysaccharide biosynthesis (i.e., starch, cellulose and callose) and respiration.^12,16 There is also evidence that SUSY improves the metabolic performance at low internal oxygen levels¹⁷ but little is known about its role during plant defense. Callose formation is presumably one of the strongest sink reactions in plant cells.^1,18 Defense-related SUSY activity may serve to allocate Suc into callose deposition and other carbohydrate-consuming defense reactions. In fact, in the resistant wt the activity of SUSY increased upon interaction with P. nicotianae in a biphasic manner (Fig. 1D). The time course is comparable to that of cwINV activity and correlates with callose deposition and enhanced respiration.^1,11 However, repression of cwINV leads in general to a reduction of SUSY activity in source leaves of tobacco.¹ After infection the activation of SUSY was also significantly impaired (Fig. 1D). At the same time, the early defense-related callose deposition in infected mesophyll cells of SNN::cwINV plants is substantially delayed.¹ It is known that expression of SUSY isoforms is differentially controlled by sugars,¹² and there is evidence that hexoses generated by the defense-induced cwINV activity deliver sugar signals to the infected cells.¹ In this sense, the reduction of defense-related, cwINV-generated sugar signals could be responsible for the repression of SUSY activity in SNN::cwINV plants after infection with P. nicotianae.Only limited hexoses or hexose-based sugar signals could be generated by cytoplasmic Suc cleavage.¹² The reduction of soluble carbohydrates for sugar signaling and also as fuel for metabolic pathways that support defense reactions could be responsible for the impaired resistance in SNN::cwINV plants (Fig. 1A).Obviously, neither intracellular INV isoforms, nor SUSY can compensate for the reduced carbohydrate availability due to cwINV repression during plant defense. The data also suggest that the activity of SUSY is affected by cwINV and related reflux of carbohydrates. It is known that SUSY activity can be controlled, e.g., by sugar-mediated phosphorylation¹² and one may speculate that posttranslational modulation of the protein is affected by the defense-related carbohydrate status of the cell.     

Sucrose Synthase: Expanding Protein Function

Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. This inference is based on the following observations: (a) tissue-specific, isoform-dependent and metabolically-regulated association of SUS with mitochondria and (b) isoform-specific and anoxia-responsive interaction of SUS with the voltage-dependent anion channel (VDAC), the major outer mitochondrial membrane protein. More recent work shows that both VDAC and SUS are also localized to the nucleus in maize seedling tissues. Their intricate regulation under anoxia indicates that these two proteins may have a role in inter-compartmental signaling.Key Words: sucrose synthase, mitochondria, nucleus, localization, voltage-dependent anion channel (VDAC), non-catalytic rolesThe biochemical function of a protein is encoded within its primary sequence and can often be deciphered by simple in vitro assays. The cellular or organismal function of a protein is frequently the same as its biochemical activity. However, for many proteins, the biological function cannot be easily derived based on its biochemical function. This appears to be particularly true when the gene encoding the protein has a history of duplication and is represented by a family of paralogs. In maize and other species, sucrose synthase (SUS) isoforms are almost identical in their catalytic properties.^1,2 However, the characteristic phenotypes of mutants in specific isoforms suggest that the isoforms contribute to vastly different organismal functions.^2–4 Our interest is to identify the range of functions that maize SUS isoforms may have and elucidate the molecular basis of this functional diversity. Although expression divergence and consequent variation in their cellular abundance significantly contributes to this diversity,⁵ other factors such as intracellular distribution, post-translational modifications and interacting partners,^3,4,6,7 seem to be equally critical for the functional diversification of different SUS isoforms.Our study, spurred by a bioinformatics prediction, opened up a new facet of SUS biology, in that the protein may have organelle-based functions.⁸ Our analysis indicated that two of the three maize SUS isoforms (SH1 and SUS1) partly localize to mitochondria and nuclei, compartments not related to sucrose metabolism. In addition to this isoform-specificity, the compartmentation of SUS isoforms is influenced by developmental as well as environmental cues. Furthermore, its isoform-specific interaction with the voltage-dependent anion channel (VDAC) and an apparent conservation of SUS mitochondrial targeting across plant species suggest that SUS may have novel, noncatalytic biological functions. Our recent work shows that along with SUS, VDAC is also localized to the nucleus and these two proteins are inversely regulated in these two compartments under anoxic stress, indicating SUS-VDAC interaction may play a role in inter-compartmental signaling (Fig. 1).Open in a separate windowFigure 1Current working model of SUS-VDAC interactions in maize root tip cells. Prolonged anoxia leads to de-oligomerization of VDAC and the release of SUS from mitochondria, resulting in the migration of SUS to the nucleus. We hypothesize that the nuclear accumulation of SUS signals the induction of cell death pathway leading to the death of the root tip in anoxic maize seedlings. The insets show the primary root tip and a part of the axis from aerobic and anoxic seedlings. The root tip death is indicated by Evans Blue staining pattern of the anoxic root. ≠ = SUS. □ = VDAC.SUS mitochondrial localization also provided us an opportunity to reinterpret the phylogeny of sucrose metabolism. The proposed origin of sucrose metabolism is equivocal between the proteobacterial and cyanobacterial lineages.^9,10 Our discovery of SUS inside mitochondria, absence of plastid-bound SUS or plastid-targeting information in any of the plant SUS proteins and occurrence of mitochondrial targeting information in proteobacterial SUS orthologs strongly support a proteobacterial origin of plant sucrose synthases.⁸ Based on a genome-wide analysis of E. coli proteins, Lucattini et al.¹¹ proposed that mitochondrial targeting information may have been derived from the preexisting sequences of the endosymbiont proteins. We hypothesize that, in addition to the structural features needed for mitochondrial association, the functional basis of SUS-VDAC interaction may have been recruited by plants from the prokaryotic SUS genes. Based on striking similarities between bacterial and mitochondrial porins in their structure as well as regulation by purine nucleotides and their role in the host-cell death as modulated by cellular ATP levels, Frade and Michaelidis¹² speculated that the eukaryotic programmed cell death may have been a consequence of acquiring aerobic metabolism via the endosymbiotic process. Is organellar SUS a part of this acquisition?     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

The Role of PP2A A Subunits in Tumor Suppression

The protein phosphatase 2A (PP2A) family of heterotrimeric serine-threonine phosphatases participates in human cell transformation. Each functional PP2A complex contains one structural A subunit (Aα or Aβ), and mutations of both are found to occur at low frequency in human tumors. We have shown that Aα functions as haploinsufficient tumor suppressor gene by regulating in part phosphatidylinositol 3-kinase (PI3K) signaling. In contrast, loss of Aβ function due to biallelic alterations contributes to cancer progression through dysregulation of small GTPase RalA activity. These observations provide evidence that dysfunction of particular PP2A complexes regulate specific phosphorylation event necessary for cancer initiation.Key Words: protein phosphatase 2A, RalA, cancer, transformationReversible phosphorylation plays a key role in the regulation of signaling pathways relevant to cell transformation. Dysregulation of several kinase oncogenes have been shown to be required for cancer development, and several targeted therapies focused on inhibiting particular kinases have now been approved for clinical use. Although it is clear that phosphorylation is also regulated by phosphatases, initial biochemical studies suggested that unlike kinases, phosphatases act promiscuously and constitutively in vitro. However, recent work indicates that phosphatases play essential roles in malignant transformation by acting on specific substrates in vivo.Protein phosphatase 2A (PP2A) is a family of serine-threonine phosphatases implicated in the control of a diverse array of cellular processes. The PP2A core enzyme consists of a catalytic C subunit and a structural A subunit. In mammals, two distinct genes encode closely related versions of both the PP2A A and C subunits. The AC dimer recruits a third regulatory B subunit that has been predicted to dictate the substrate specificity and function of the PP2A heterotrimeric complex. Four unrelated families of B subunits have identified to date: B/B55/PR55/PPP2R2, B′/B56/PR61/PPP2R5, B″/PR72/PPP2R3 and Striatin¹ (Fig. 1). Recent genetic and proteomic studies implicate clear roles for PP2A subunits in regulating physiological functions and one emerging view is that specific PP2A complexes play critical roles in cell transformation by regulating particular substrates.Open in a separate windowFigure 1Disruption of PP2A complexes induces transformation. PP2A is a heterotrimeric protein complex, and several isoforms exist for each of the three subunits, creating a diverse family of related enzymes that regulate specific physiological functions. Alterations of PP2A structural subunits, Aα and Aβ, contribute to spontaneously arising human cancers by distinct mechanisms. Cancer-associated Aα haploinsufficiency may induce human cell transformation by activating PI3K/AKT pathway while PP2A Aβ loss-of-function permits the accumulation of activated RalA.Somatic alterations of the PP2A structural subunit Aβ (PPP2R1B) have been found to occur in colon, lung and breast cancers.^2–5 Notably, point mutations in one Aβ allele are commonly accompanied by loss of the second Aβ allele. We confirmed previous work⁶ that showed cancer-associated Aβ mutants form functionally null alleles.⁷ These studies indicate that Aβ is genetically inactivated in a subset of human cancers. In addition, we found that suppression of Aβ was found to cooperate with H-Ras, telomerase catalytic subunit hTERT and the SV40 Large T antigen to induce transformation of normal human cells while introduction of wild type Aβ into lung carcinoma cells lacking functional Aβ partially reverses this tumorigenic phenotype.⁷ Together, these data provide evidence that PP2A Aβ functions as a tumor suppressor gene.Previous work has shown cancer derived Aβ mutants exhibit markedly impaired ability to form complexes with the catalytic C subunit and the regulatory PR72 subunit.⁶ We have found that Aβ mutants also showed decreased ability to bind to regulatory Bα subunit and several members of B′ family. These data indicate that cancer-associated alterations of PP2A Aβ result in disruption of most if not all PP2A Aβ-containing complexes. Considering that distinct Aβ-B complexes are likely regulate the phosphorylation of particular substrates involved in transformation, further work is required to identify which B subunits participate in malignant transformation.Somatic mutations of the more abundant PP2A structural Aα subunit have also been reported in human cancers, although at low frequency.^2,8 We previously showed that cancer-associated PP2A Aα mutations contribute to cell transformation by creating a state of haploinsufficiency.⁹ Although these two distinct PP2A structural isoforms, Aα and Aβ, are 86% identical,¹⁰ it was unclear whether these two isoforms share overlapping functions.¹¹ We found that overexpression of Aα failed to revert the tumorigenic phenotype induced by Aβ suppression, suggesting that PP2A complexes containing Aα or Aβ are functionally distinct.To identify substrates specific for PP2A Aβ, we performed large scale immunopurification of PP2A Aα- and Aβ-containing complexes. We have found that PP2A Aβ complex, but not the PP2A Aα complex, binds to and inhibits activity of the small GTPase RalA through direct dephosphorylation at Ser183 and Ser 194. Cancer-associated Aβ mutants are unable to dephosphorylate RalA, suggesting that loss of Aβ function impairs the formation of complexes with RalA and deregulates its activity. Consistent with previous reports that implicated RalA in regulation of several signaling pathways relevant to cell transformation,^12–14 loss of function experiments revealed that RalA is crucial for transformation mediated by Aβ dysfunction. These findings strongly suggest that accumulation of phospho-RalA in PP2A Aβ deficient cells promotes tumorigenic phenotype (Fig. 1). However, we cannot exclude that other substrates of PP2A Aβ complexes also contribute to cell transformation.These observations also implicate phosphorylation of RalA as an alternative mechanism that may regulate RalA activity and cell transformation. Prior work has shown Aurora A kinase as one kinase that can induce RalA phosphorylation at Ser 194.¹⁵ However, further studies are required to identify the kinase(s) that are responsible for RalA phosphorylation at Ser 183 and Ser 194.While Aβ loss-of-function permits the accumulation of activated RalA, Aα haploinsufficiency seems to induce human cell transformation by activating AKT/PI3K signaling pathway⁹ (Fig. 1). However, it remains unclear whether PP2A A subunits determine the substrate specificity of heterotrimeric complexes by direct substrate binding, or by forming complex with particular set of B and C subunits. In consonance with the latter idea, Aα and Aβ have been reported to have different affinity to Cα, Bα, B''α1 and PR72 subunits.¹⁷ The systematic characterization of PP2A complex composition necessary for RalA dephosphorylation and Akt activation and further structural studies to resolve PP2A in complex with specific substrates will help elucidate the mechanistic details of how PP2A acts as a tumor suppressor.     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

v-Crk regulates membrane dynamics and Rac activation

Cell migration is an integrated process that involves cell adhesion, protrusion and contraction. We recently used CAS (Crk-associated substrate, 130CAS)-deficient mouse embryo fibroblasts (MEFs) to examined contribution made to v-Crk to that process via its interaction with Rac1. v-Crk, the oncogene product of avian sarcoma virus CT10, directly affects membrane ruffle formation and is associated with Rac1 activation, even in the absence of CAS, a major substrate for Crk. In CAS-deficient MEFs, cell spreading and lamellipodium dynamics are delayed; moreover, Rac activation is significantly reduced and it is no longer targeted to the membrane. However, expression of v-Crk by CAS-deficient MEFs increased cell spreading and active lamellipodium protrusion and retraction. v-Crk expression appears to induce Rac1 activation and its targeting to the membrane, which directly affects membrane dynamics and, in turn, cell migration. It thus appears that v-Crk/Rac1 signaling contributes to the regulation of membrane dynamics and cell migration, and that v-Crk is an effector molecule for Rac1 activation that regulates cell motility.Key words: v-Crk, Rac, lamellipodia dynamics, cell migration, p130CASCell migration is a central event in a wide array of biological and pathological processes, including embryonic development, inflammatory responses, angiogenesis, tissue repair and regeneration, cancer invasion and metastasis, osteoporosis and immune responses.^1,2 Although the molecular basis of cell migration has been studied extensively, the underlying mechanisms are still not fully understood. It is known that cell migration is an integrated process that involves formation of cell adhesions and/or cell polarization, membrane protrusion in the direction of migration (e.g., filopodium formation and lamellipodium extension), cell body contraction and tail detachment.^1–3 Formation of cell adhesions, including focal adhesions, fibrillar adhesions and podosomes are the first step in cell migration. Cell adhesions are stabilized by attachment to the extracellular matrix (ECM) mediated by integrin transmembrane receptors, which are also linked to various cytoplasmic proteins and the actin cytoskeleton, which provide the mechanical force necessary for migration.^2,4 The next steps in the process of cell migration are filopodium formation and lamellipodium extension. These are accompanied by actin polymerization and microtubule dynamics, which also contribute to the control of cell adhesion and migration.⁵Focal adhesions are highly dynamic structures that form at sites of membrane contact with the ECM and involve the activities of several cellular proteins, including vinculin, focal adhesion kinase (FAK), Src family kinase, paxillin, CAS (Crk-associated substrate, p130CAS) and Crk.⁶ A deficiency in focal adhesion protein is associated with the severe defects in cell motility and results in embryonic death. For example, FAK deficiency disrupts mesoderm development in mice and delays cell migration in vitro,⁷ which reflects impaired assembly and disassembly the focal adhesions.⁸ In addition, mouse embryonic fibroblasts (MEFs) lacking Src kinase showed a reduced rate of cell spreading that resulted in embryonic death.⁹ Taken together, these findings strongly support the idea that cell adhesion complexes play crucial roles in cell migration.CAS is a hyperphosphorylated protein known to be a major component of focal adhesion complexes and to be involved in the transformation of cells expressing v-Src or v-Crk.¹⁰ CAS-deficient mouse embryos die in utero and show marked systematic congestion and growth retardation,⁴ while MEFs lacking CAS show severely impaired formation and bundling of actin stress fibers and delayed cell motility.^4,11,12 Conversely, transient expression of CAS in COS7 cells increases cell migration.¹¹ Crk-null mice also exhibit lethal defects in embryonic development,¹³ which is consistent with the fact that CAS is a major substrate for v-Crk, and both CAS and v-Crk are necessary for induction of cell migration.¹⁴ v-Crk consists of a viral gag sequence fused to cellular Crk sequences, which contain Src homology 2 (SH2) and SH3 domains but no kinase domain, and both CAS and paxillin bind to SH2 domains.^12,15,16 Despite the absence of a kinase domain, cell expressing v-Crk show upregulation of tyrosine phosphorylation of CAS, FAK and paxillin, which is consistent with v-Crk functioning as an adaptor protein.¹⁷ Moreover, this upregulation of tyrosine phosphorylation correlates well with the transforming activity of v-Crk.¹⁷ By contrast, tyrosine phosphorylation of FAK and CAS is diminished in Src kinase-deficient cells expressing v-Crk, and they are not targeted to the membrane, suggesting v-Crk signaling is Src kinase-dependent. After formation of the CAS/v-Crk complex, v-Crk likely transduces cellular signaling to Src kinase and FAK.¹² Notably, tyrosine phosphorylation of FAK and cell migration and spreading are all enhanced when v-Crk is introduced into CAS-deficient MEFs.¹² We therefore suggest that v-Crk activity, but not cellular Crk activity, during cell migration and spreading is CAS-independent.Membrane dynamics such as lamellipodium protrusion and membrane ruffling reportedly involve Rac1,¹⁸ α4β1 integrin,¹⁹ Arp2/3,⁶ and N-WASP,²⁰ and are enhanced in v-Crk-expressing CAS-deficient MEFs.²¹ Moreover, expression in those cells of N17Rac1, a dominant defective Rac1 mutant, abolished membrane dynamics at early times and delayed cell migration.²¹ v-Crk-expressing, CAS-deficient MEFs transfected with N17Rac1 did not begin spreading until one hour after being plated on fibronectin, and blocking Rac activity suppressed both membrane dynamics and cell migration. We therefore suggest that v-Crk is involved in cell attachment and spreading, and that this process is mediated by Rac1 activation. In addition, v-Crk expression apparently restores lamellipodium formation and ruffle retraction in CAS-deficient MEFs. Thus v-Crk appears to participate in a variety cellular signaling pathways leading to cell spreading, Rac1 activation, membrane ruffling and cell migration, even in the absence of CAS, its major substrate protein.In fibroblasts, the Rho family of small GTP-binding proteins (e.g., Cdc42, Rac and Rho) functions to control actin cytoskeleton turnover, including filopodium extension, lamellipodium formation and generation of actin stress fibers and focal adhesions.²² These GTPases function in a cascade, such that activation of Cdc42 leads to activation of Rac1, which in turn activates Rho.²² Once activated, Rho controls cell migration. Cell adhesion to ECM leads to the translocation of Rac1 and Cdc42 from the cytosol to the plasma membrane,²³ where they regulate actin polymerization at the leading edge.^19,24 Dominant negative Rac and Cdc42 mutants inhibit the signaling to cell spreading initiated by the interaction of integrin with ECM.²⁴ The fact that cellular levels of activated Rac are higher in cells adhering to ECM than in suspended cells further suggests that activation of Rac and Cdc42 is a critical step leading to membrane protrusion and ruffle formation. It is noteworthy in this regard that v-Crk is able to induce Rac activation and its translocation to plasma membrane.²¹Overall, the findings summarized in this article demonstrate that v-Crk participates in several steps leading to cell adhesion and spreading (Fig. 1), and the targeting of v-Crk to focal adhesion sites appears to be a prerequisite for regulation of cell migration and spreading via Rac activation. To fully understand its function, however, it will be necessary to clarify the role of v-Crk in Rac1 and Cdc42 activation initiated by integrin-ECM interactions.Open in a separate windowFigure 1Schematic diagram of v-Crk signaling in MEFs. Cell adhesion signaling initiated by the integrin-ECM interaction triggers v-Crk signaling mediated by Src kinase, after which focal adhesion proteins are tyrosine phosphorylated. These events lead to translocation of Rac from the cytosol to the membrane, where it promotes membrane protrusion and ruffle formation. Under basal conditions, Rac is bound with GDP and is inactive. Upon stimulation, Rac activation is mediated by guanine nucleotide exchange factors (GEFs) that stimulate the release of bound GDP and the binding of GTP. Activation of Rac is transient, however, as it is inactivated by GTPase activating protein (GAP).     

Molecular functions of the PP2A regulatory subunit Tap46 in plants

Tap42/α4 is a regulatory subunit of the protein phosphatase 2A (PP2A) family of phosphatases and plays a role in the target of rapamycin (TOR) pathway that regulates cell growth, ribosome biogenesis, translation and cell cycle progression in both yeast and mammals. We determined the cellular functions of Tap46, the plant homolog of Tap42/α4, in both Arabidopsis thaliana and Nicotiana benthamiana. Tap46 associated with the catalytic subunits of PP2A and the PP2A-like phosphatases PP4 and PP6 in vivo. Tap46 was phosphorylated by TOR in vitro, indicating that Tap46 is a direct substrate of TOR kinase. Tap46 deficiency caused cellular phenotypes that are similar to TOR-depletion phenotypes, including repression of global translation and activation of both autophagy and nitrogen recycling. Furthermore, Tap46 depletion regulated total PP2A activity in a time-dependent manner similar to TOR deficiency. These results suggest that Tap46 acts as a positive effector of the TOR signaling pathway in controlling diverse metabolic processes in plants. However, Tap46 silencing caused acute cell death, while TOR silencing only hastened senescence. Furthermore, mitotic cells with reduced Tap46 levels exhibited chromatin bridges at anaphase, while TOR depletion did not cause a similar defect. These findings suggest that Tap46 may have TOR-independent functions as well as functions related to TOR signaling in plants.Key words: acute cell death, autophagy, chromatin bridge, nitrogen mobilization, protein phosphatases, target of rapamycin (TOR)Yeast type 2A phosphatase-associated protein 42 kDa (Tap42) is a regulatory subunit that directly associates with catalytic subunits of the protein phosphatase 2A (PP2A) family of protein phosphatases to make a heterodimer and regulates the activity and substrate specificity of the intact enzyme complex.¹ Functions of Tap42 as a component of the target of rapamycin (TOR) signaling pathway have been well characterized in yeast.^1–3 Tap42-regulated phosphatase activities play a major role in signal transduction mediated by TOR. Accumulating evidence suggest that TOR regulates phosphorylation of target proteins by restraining PP2A activity through Tap42 phosphorylation.^1–3 Rapamycin inhibits TOR activity and also influences Tap42-mediated phosphatase regulation in yeast.^3–5α4, the mammalian homolog of Tap42, also associates with the catalytic subunits of PP2A, PP4 and PP6 to make a heterodimer.⁶ Rapamycin inhibits mammalian TOR (mTOR) activity, but it is not clear whether rapamycin prevents the formation of the α4/PP2Ac complex or whether α4 stimulates or represses PP2Ac activity.^7–9 Interestingly, loss of Tap42 function in Drosophila does not affect TOR-regulated activities, including cell growth, metabolism and S6 kinase activity, but results in mitotic arrest caused by spindle anomalies and subsequent activation of c-Jun N-terminal kinase signaling and apoptosis.¹⁰ Similarly, α4 deletion in mice leads to the rapid onset of apoptosis in both proliferating and differentiated cells, while rapamycin itself does not severely affect adult cells.¹¹ Furthermore, while TOR depletion causes developmental arrest and organ degeneration at the L3 stage in Caenorhabditis elegans, loss of α4 does not reproduce TOR deficiency phenotypes, but mainly leads to a fertility defect.¹² Taken together, these results suggest that the yeast Tap42/TOR paradigm is not completely conserved in higher eukaryotes and that Tap42/α4 functions may not be exclusively dependent on the Tor signaling pathway.In this study, we investigated the in vivo functions and phosphatase regulation of Tap46, the plant Tap42/α4 homolog, in relation to TOR in Nicotiana benthamiana, Arabidopsis and tobacco BY2 cells. Tap46 was shown to interact with the catalytic subunits of PP2A, PP4 and PP6 in vivo. Recombinant Tap46 protein was phosphorylated by immunoprecipitated TOR kinase and its deletion forms in vitro. Dexamethasone-induced RNAi of Tap46 caused dramatic repression of global translation and activation of both autophagy and nitrogen mobilization in the early stages of gene silencing. These phenotypes mimic those of TOR inactivation or TOR deficiency in Arabidopsis, yeast and mammals, indicating that Tap46 is a critical mediator of the Tor pathway in the regulation of these metabolic processes in plants. However, these early phenotypes of Tap46-deficient plants were soon followed by an acute and rapid programmed cell-death (PCD), while TOR silencing only led to growth retardation and premature senescence in Arabidopsis and N. benthamiana, confirming results from a previous study.¹³ The PCD caused by Tap46 deficiency is consistent with the apoptosis induced by loss of Tap42/α4 function in both Drosophila and mice.^10,11 Thus Tap42/α4/Tap46 appears to have a strong anti-apoptotic activity in higher eukaryotes. The underlying mechanisms of PCD activation caused by Tap46 depletion remain to be revealed, but it is possible that the inappropriate modulation of phosphatase activity and aberrant protein phosphorylation led to stress signaling and PCD activation.Another interesting phenotype of Tap46 deficiency is the formation of chromatin bridges in anaphase during mitosis, suggesting a role for Tap46 in plant cell mitotic progression. However, there have been no reports of anaphase bridge formation in tor mutants of any organisms. In Drosophila, loss of Tap42 function causes spindle disorganization and pre-anaphase arrest prior to the onset of apoptosis.¹⁰ In addition, Drosophila mutants with a defective regulatory subunit of PP2A exhibit an increased number of lagging chromosomes and chromatin bridges in anaphase.^14,15 Tap46 likely regulates the functions of PP2A family phosphatases during mitosis by direct association with their catalytic subunits, thereby modulating both the activity and specificity of the enzyme. Accumulating evidence reveals dynamic functions of PP2A during mitosis in both yeast and mammals: PP2A regulates kinetochore function, sister chromatid cohesion, spindle bipolarity and progression to anaphase.^15–17 Counteracting the activity of protein kinases, PP4 has also been implicated in both centrosome maturation and function during mitosis.¹⁸ Based on immunolabeling results, Tap46 was visualized as distinct spots around chromatin and mitotic spindles during mitosis in tobacco BY2 cells (Lee HS and Pai HS, unpublished results). Further studies will address the interacting partners and dynamic relocation of Tap46 during the cell cycle.Our results in this study demonstrated that Tap46 plays an important regulatory role in plant growth and metabolism; a major part of its function appears related to TOR signaling. However, we consistently observed certain phenotypic differences between Tap46-silenced and TOR-silenced Arabidopsis and N. benthamiana plants: an acute and rapid PCD occurred upon Tap46 silencing but not upon TOR silencing, despite a similar degree of gene silencing. Furthermore, we did not observe anaphase bridge formation in mitotic root-tip cells of ethanol-induced TOR RNAi Arabidopsis plants, while chromatin bridges were repeatedly observed in Tap46-silenced tobacco BY2 and Arabidopsis root-tip cells. Although an ancient Tap42/TOR paradigm observed in yeast appears to be conserved in plants, new TOR-independent functions of Tap46 might have evolved, the abrogation of which can cause massive PCD activation and anaphase bridge formation. Tap46 is a major regulator of cellular PP2A activity in plant cells by interacting with multiple phosphatase partners. Unraveling the molecular networks of Tap46 activity and interactions is essential for understanding its TOR-dependent and -independent functions in plants.     

Putting the brakes on cancer cell migration: JAM-A restrains integrin activation

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, downregulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and downregulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.Key words: JAM-A, integrin, α_vβ₃, FGF-2, breast cancer, cell migration and invasion, T47D, MDA-MB-231, siRNAEndothelial and epithelial cells exhibit cell polarity and have characteristic tight junctions (TJs) that separate apical and basal surfaces. TJs are composed of both transmembrane and cytoplasmic proteins. The three major families of transmembrane proteins include claudins, occludin and JAM family members.^1–3 Additionally, interaction between the peripheral proteins such as PDS-95/Discs large/ZO family (PDZ) domain-containing proteins in TJs plays an important role in maintaining the junctional integrity.^2,4,5JAMs are type I membrane proteins (Fig. 1) predominately expressed in endothelial and epithelial cell TJs, platelets and some leukocytes.^6–8 The classical JAMs are JAM-A, JAM-B and JAM-C, which can all regulate leukocyte-endothelial cell interaction through their ability to undergo heterophilic binding with integrins α_Lβ₂ or α_vβ₃, α₄β₁ and α_Mβ₂ respectively. The cytoplasmic tail of JAMs contains a type II PDZ-domain-binding motif (Fig. 1) that can interact with the PDZ domain containing cytoplasmic molecules such as ZO-1, ASIP/PAR-3 or AF-6.^9,10 Additionally, consistant with their junctional localization and their tendency to be involved in homophilic interactions, JAMs have been shown to modulate paracellular permeability and thus may play an important role in regulating the epithelial and endothelial barrier.^11,12 In addition, ectopic expression of JAM-A in CHO cells promotes localization of ZO-1 and occludin at points of cell contacts, which suggests a role for JAM-A in TJ assembly.^10,13,14 Recently, it has been shown that JAM-A regulates epithelial cell morphology by modulating the activity of small GTPase Rap1 suggesting a role for JAM-A in intracellular signaling.¹⁵Open in a separate windowFigure 1Schematic representation of the domain structure of JAM family proteins. V, variable Ig domain; C2, constant type 2 Ig domain; TM, transmembrane domain; T-II, Type II PDZ-domain binding motif.We have previously shown that JAM-A is a positive regulator of fibroblast growth factor-2 (FGF-2) induced angiogenesis.¹⁶ Evidence was provided to support the notion that JAM-A forms a complex with integrin α_vβ₃ at the cell-cell junction in quiescent human umbilical cord vein endothelial cells (HUVECs) and FGF-2 dissociates this complex.¹⁶ It was further established that inhibition of JAM-A using a function-blocking antibody also inhibits FGF-2 induced HUVECs migration in vitro and angiogenesis in vivo. Overexpression of JAM-A induced a change in HUVECs morphology similar to that observed when treated with FGF-2.¹⁷ Furthermore, overexpression of JAM-A, but not its cytoplasmic domain deletion mutant, augmented cell migration in the absence of FGF-2.¹⁷ In addition, downregulation of JAM-A in HUVECs using specific siRNA, resulted in reduced FGF-2-induced cell migration and inhibition of mitogen activated protein (MAP) kinase activation.¹⁸ These findings clearly suggested that JAM-A positively regulates FGF-2-induced endothelial cell migration. This was further confirmed in vivo by using JAM-A null mouse in which FGF-2 failed to support angiogenesis.¹⁹It is known that JAM-C, a JAM family member, is involved in the process of tumor cell metastasis.²⁰ However, little is known about JAM-A''s role in cancer progression. We recently found that JAM-A is expressed in breast cancer tissues and cell lines.²¹ Based on our studies with endothelial cells it was felt that JAM-A expression in breast cancer cells may also enhance the migratory ability of these cells. Surprisingly, we found an inverse relation between the expression of JAM-A and the metastatic ability of breast cancer cells. T47D cells, which express high levels of JAM-A, are the least migratory; whereas MDA-MB-231 cells, which are highly migratory, are found to express the least amount of JAM-A.²¹ We also found that overexpression of JAM-A in MDA-MB-231 cells caused a change in cell morphology from spindle-like to rounded shape and formed cobblestone-like clusters.²¹ This is consistent with the previous report, that downregulation of JAM-A expression from epithelial cells using siRNA results in the change of epithelial cell morphology.¹⁵ This change in cell morphology by knockdown of JAM-A was attributed to the disruption of epithelial cell barrier function.¹⁵ It was further shown that knockdown of JAM-A affects epithelial cell morphology through reduction of β₁integrin expression due to decreased Rap1 activity.¹⁵ Our observed effect of JAM-A downregulation in T47D cells, however, is not due to downregulation of β₁integrin, since the level of this integrin was not affected in these cells. Interestingly, overexpression of JAM-A significantly affected both the cell migration and invasion of MDA-MB-231 cells. Furthermore, knockdown of JAM-A using siRNA enhanced invasiveness of MDA-MB-231 cells, as well as T47D cells.²¹ The ability of JAM-A to attenuate cell invasion was found to be due to the formation of functional tight junctions as observed by distinct accumulation of JAM-A and ZO-1 at the TJs and increased transepithelial resistance. These results identify, for the first time, a tight junctional cell adhesion protein as a key negative regulator of breast cancer cell migration and invasion.²¹JAM-A has been shown to be important in maintaining TJ integrity.^15,22–25 Disruption of TJs has been implicated to play a role in cancer cell metastasis by inducing epithelial mesenchymal transition.²⁶ Several laboratories, including ours, have shown that cytokines and growth factors redistribute JAM-A from TJs.^16,27,28 Consistent with this finding, it has been shown that hepatocyte growth factor (HGF) disrupts TJs in human breast cancer cells and downregulates expression of several TJ proteins.²⁹ It is therefore conceivable that the loss of JAM-A in highly metastatic cells is a consequence of disruption of TJs. This was further supported by the findings that overexpression of JAM-A forms functional TJs in MDA-MB-231 cells and attenuates their migratory behavior. Our result is the first report correlating an inverse relationship of JAM-A expression in breast cancer cells to their invasive ability.²¹Using cDNA microarray technology, it has been revealed how genes involved in cell-cell adhesion, including those of the TJ, are under or overexpressed in different carcinomas.^15,30 Cell-cell adhesion molecules have been well documented to regulate cancer cell motility and invasion. Of these, the cadherin family have been studied the most.^31,32 It was proposed that a cadherin switch, that is, the loss of E-cadherin and subsequent expression of N-cadherin, may be responsible for breast cancer cell invasion.^33,34 Although the role of cadherins is well-documented, it remains controversial since some breast cancer cell lines that do not express these proteins still posses highly invasive characteristics.^33,34 However, the observed effect of overexpression of JAM-A does not appear to be simply due to the formation of TJs, since individual cells that express increased JAM-A show reduced migration.²¹ This is not surprising, considering the fact that JAM-A in addition to its function of regulating TJ integrity is also shown to participate in intracellular signaling. JAM-A is capable of interacting homotypically as well as heterotypically on the cell surface.^35,36 It has also been shown that it interacts with several cytoplasmic proteins through its PDZ domain-binding motif and recruits signaling proteins at the TJs.³⁷ Recent findings using site-directed mutagenesis suggest that cis-dimerization of JAM-A is necessary for it to carry out its biological functions.³⁸ Our own observations suggest that a JAM-A function-blocking antibody inhibits focal adhesion formation in endothelial cells (unpublished data), whereas overexpresion of JAM-A in MDA-MB-231 cells show increased and stable focal adhesions.²¹ It is therefore conceivable that in quiescent endothelial/epithelial cells JAM-A associates with integrin to form an inactive complex at the TJ (Fig. 2). Growth factors such as FGF-2 signaling dissociates this complex thus allowing dimerization of JAM-A and activation of integrin augmenting cell migration (Fig. 2). On the contrary, in MDA-MB-231 cancer cells, which express low levels of JAM-A and do not form tight junctions, there may not be efficient inactive complex formation between JAM-A and integrin. Overexpression of JAM-A in these cells however, may promote such inactive complex formation leading to inhibition of integrin activation and JAM-A dimerization, both necessary events for cell migration. We are currently in the process of determining the specificity of interaction of JAM-A with integrins. Further experimentation is ongoing to determine the contribution of JAM-A dependent signaling in cell migration.Open in a separate windowFigure 2Schematic representation of JAM-A regulation of cell migration. JAM-A forms an inactive complex with the integrin and sequesters it at the TJs. Growth factor signaling dissociates this complex, promoting integrin activation and JAM-A dimerization leading to cell migration via MAP kinase activation. Ectopic expression of JAM-A in cancer cells may induce its association with integrin, forming an inactive complex and hence attenuation of migration.JAM-A differentially regulates cell migration in endothelial and cancer cells due to its ability to form inactive complex with integrin, making it a metastasis suppressor. The downregulation of JAM-A in carcinoma cells may be detrimental to the survival of breast cancer patients. It is therefore very important to determine the molecular determinants that are responsible for the downregulation of JAM-A during cancer progression. Thus, JAM-A, a molecule that dictates breast cancer cell invasion, could be used as a prognostic marker for metastatic breast cancer.