Regulation of protein kinase C-related protein kinase 2 (PRK2) by an intermolecular PRK2-PRK2 interaction mediated by Its N-terminal domain

Protein kinase C-related protein kinases (PRKs) are effectors of the Rho family of small GTPases and play a role in the development of diseases such as prostate cancer and hepatitis C. Here we examined the mechanism underlying the regulation of PRK2 by its N-terminal region. We show that the N-terminal region of PRK2 prevents the interaction with its upstream kinase, the 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates the activation loop of PRK2. We confirm that the N-terminal region directly inhibits the kinase activity of PRK2. However, in contrast to previous models, our data indicate that this inhibition is mediated in trans through an intermolecular PRK2-PRK2 interaction. Our results also suggest that amino acids 487-501, located in the linker region between the N-terminal domains and the catalytic domain, contribute to the PRK2-PRK2 dimer formation. This dimerization is further supported by other N-terminal domains. Additionally, we provide evidence that the region C-terminal to the catalytic domain intramolecularly activates PRK2. Finally, we discovered that the catalytic domain mediates a cross-talk between the inhibitory N-terminal region and the activating C-terminal region. The results presented here describe a novel mechanism of regulation among AGC kinases and offer new insights into potential approaches to pharmacologically regulate PRK2.     

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer

In humans, thromboxane (TX) A(2) signals through the TPα and TPβ isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.     

Inhibition of Akt and its anti-apoptotic activities by tumor necrosis factor-induced protein kinase C-related kinase 2 (PRK2) cleavage

Akt is stimulated by several growth factors and has a major anti-apoptotic role in the cell. Therefore, we hypothesized that a pathway leading to the inhibition of Akt might be utilized in the process of apoptosis. Accordingly, we used a yeast two-hybrid screening assay to identify the proteins that interact with and possibly inhibit Akt. We found that the C-terminal region of protein kinase C-related kinase 2 (PRK2), containing amino acids 862 to 908, specifically binds to Akt in yeast and mammalian cells. During early stages of apoptosis, the C-terminal region of PRK2 is cleaved from the inhibitory N-terminal region and can bind Akt. The protein-protein interaction between Akt and the PRK2 C-terminal region specifically down-modulates the protein kinase activities of Akt by inhibiting phosphorylation at threonine 308 and serine 473 of Akt. This inhibition of Akt leads to the inhibition of the downstream signaling of Akt in vivo. The PRK2 C-terminal fragment strongly inhibits the Akt-mediated phosphorylation of BAD, a pro-apoptotic Bcl-2 family protein, and blocks the anti-apoptotic activities of Akt in vivo. These results provide direct evidence that the products of protein cleavage during apoptosis inhibit pro-survival signalings, leading to the amplification of pro-apoptotic signalings in the cell.     

Activation of the small GTPase Rac1 by cGMP-dependent protein kinase

Cyclic-GMP-dependent protein kinase (PKG) is widely appreciated as having diverse roles in a variety of cell types. Many reports have indicated that PKG might regulate cell function by activating members of the mitogen-activated protein kinase (MAPK) family of signaling proteins. In this study, stimulation of HEK-293 cells with nitric oxide (NO) was found to induce a rapid accumulation of phosphorylated p38 MAPK. The involvement of PKG in this process was confirmed by cotransfection of a dominant negative PKG construct (G1alphaR-GFP), which was able to block cGMP-induced p38 MAPK activation. Transfection of cells to express dominant negative Rac1(T17N) was also able to dose-dependently block cGMP-stimulated activation of p38 MAPK, thus indicating the importance of this pathway downstream of PKG. GST-PDB affinity-precipitation experiments revealed that stimulation of HEK293 cells with either nitric oxide or 8-Br-cGMP resulted in a rapid and transient activation of Rac1 with similar kinetics to p38 MAPK phosphorylation. Moreover, using in vitro kinase assays it was found that cGMP also stimulated the activity of the Rac1 effector Pak1. The activation of both Rac1 and Pak1 by 8-Br-cGMP was completely abolished by transfection of the cells with G1alphaR-GFP. Expression of the Rac1(T17N) mutant inhibited PKG-dependent activation of PAK1 indicating that Rac1 functions upstream of PAK1 in this pathway. Immunofluorescence experiments demonstrated clear colocalization of PKG and Rac1 in membrane ruffles and dynamic membrane regions supporting a functional interaction. However, in vitro kinase assays demonstrated that Rac1 is not a substrate for PKG suggesting an indirect activation mechanism. Taken together these data demonstrate a novel PKG-dependent pathway by which the Rac1/Pak1 pathway is activated. Furthermore, we demonstrate that this pathway is central to the activation of p38 MAPK by PKG in these cells.     

Structural basis for small G protein effector interaction of Ras-related protein 1 (Rap1) and adaptor protein Krev interaction trapped 1 (KRIT1)

Cerebral cavernous malformations (CCMs) affect 0.1–0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Å co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain “modules.”     

The protein kinase C-related kinase PRK2 interacts with the protein tyrosine phosphatase PTP-BL via a novel PDZ domain binding motif

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.     

Regulation of mitogen-activated protein kinases in cardiac myocytes through the small G protein Rac1

Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.     

Activation of protein kinase D3 by signaling through Rac and the alpha subunits of the heterotrimeric G proteins G12 and G13

PKD is the founding member of a novel protein kinase family that also includes PKD2 and PKD3. PKD has been the focus of most studies up to date, but little is known about the mechanisms that mediate PKD3 activation. Here, we show that addition of aluminum fluoride to COS-7 cells cotransfected with PKD3 and Galpha13 or Galpha12 induced PKD3 activation, which was associated with a transient plasma membrane translocation of cytosolic PKD3. Treatment with Clostridium difficile toxin B blocked PKD3 activation induced by either bombesin or by aluminum fluoride-stimulated Galpha12/13 but did not affect Galphaq-induced PKD3 activation. Furthermore, PKD3 immunoprecipitated from cells cotransfected with a constitutively active Rac (RacV12) exhibited a marked increase in PKD3 basal catalytic activity. In contrast, cotransfection with active Rho (RhoQ63L), Cdc42 (Cdc42Q61L), or Ras (RasV12) did not promote PKD3 activation. Expression of either COOH-terminal dominant-negative fragment of Galpha13 or dominant negative Rac (Rac N17) attenuated bombesin-induced PKD3 activation. Treatment with protein kinase C (PKC) inhibitors prevented the increase in PKD3 activity induced by RacV12 and aluminum fluoride-stimulated Galpha12/13. The catalytic activation of PKD3 in response to RacV12, alpha12/13 signaling or bombesin correlated with Ser-731/Ser-735 phosphorylation in the activation loop of this enzyme. Our results indicate that Galpha12/13 and Rac are important components in the signal transduction pathways that mediate bombesin receptor-induced PKD3 activation.     

Rac1 protein rescues neurite retraction caused by G2019S leucine-rich repeat kinase 2 (LRRK2)

Mutations in leucine-rich repeat kinase 2 (LRRK2) are currently the most common genetic cause of familial late-onset Parkinson disease, which is clinically indistinguishable from idiopathic disease. The most common pathological mutation in LRRK2, G2019S LRRK2, is known to cause neurite retraction. However, molecular mechanisms underlying regulation of neurite length by LRRK2 are unknown. Here, we demonstrate a novel interaction between LRRK2 and the Rho GTPase, Rac1, which plays a critical role in actin cytoskeleton remodeling necessary for the maintenance of neurite morphology. LRRK2 binds strongly to endogenous or expressed Rac1, while showing weak binding to Cdc42 and no binding to RhoA. Co-expression with LRRK2 increases Rac1 activity, as shown by increased binding to the p21-activated kinase, which modulates actin cytoskeletal dynamics. LRRK2 constructs carrying mutations that inactivate the kinase or GTPase activities do not activate Rac1. Interestingly, LRRK2 does not increase levels of membrane-bound Rac1 but dramatically changes the cellular localization of Rac1, causing polarization, which is augmented further when LRRK2 is co-expressed with constitutively active Rac1. Four different disease-related mutations in LRRK2 altered binding to Rac1, with the G2019S and R1441C LRRK2 mutations attenuating Rac1 binding and the Y1699C and I2020T LRRK2 mutations increasing binding. Co-expressing Rac1 in SH-SY5Y cells rescues the G2019S mutant phenotype of neurite retraction. We hypothesize that pathological mutations in LRRK2 attenuates activation of Rac1, causing disassembly of actin filaments, leading to neurite retraction. The interactions between LRRK2 and Rho GTPases provide a novel pathway through which LRRK2 might modulate cellular dynamics and contribute to the pathophysiology of Parkinson disease.     

Regulation of the Interaction between Protein Kinase C-related Protein Kinase 2 (PRK2) and Its Upstream Kinase, 3-Phosphoinositide-dependent Protein Kinase 1 (PDK1)

The members of the AGC kinase family frequently exhibit three conserved phosphorylation sites: the activation loop, the hydrophobic motif (HM), and the zipper (Z)/turn-motif (TM) phosphorylation site. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates the activation loop of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by ³²P_i revealed phosphorylation at two sites, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do not apply for other AGC kinases.The regulation of protein function by phosphorylation and dephosphorylation is a key mechanism of intracellular signaling pathways in eukaryotic organisms. Protein phosphorylation is catalyzed by protein kinases, which are themselves often regulated by phosphorylation (1). The specificity of protein kinases is essential for their cellular functions. In some groups of protein kinases, the specificity is achieved by means of “docking interactions.” Protein kinase docking interactions involve a recognition site on the kinase or a flanking domain that is different from the active site. The most notable example, MAP kinases, uses a docking interaction to specifically recognize substrates, upstream kinases, and phosphatases. Despite the large amount of data on protein kinase docking interactions, e.g. in the MAP kinase field, there is very little information on how these essential interactions are regulated (2–4).3-Phosphoinositide-dependent protein kinase 1 (PDK1)³ belongs to the AGC family of protein kinases and is the activation loop kinase for several other AGC kinases (5). A key feature of the AGC kinase family members except PDK1 is the presence of a C-terminal extension (CT) to the catalytic core that contains a conserved hydrophobic motif (HM) harboring a phosphorylation site. In many AGC kinases, the HM mediates a docking interaction with PDK1. For example, p90 ribosomal S6 kinase (RSK), p70 S6 kinase (S6K) and serum- and glucocorticoid-induced protein kinase (SGK) interact with PDK1 upon phosphorylation of the HM site (6–9). The phosphorylated HM binds to a HM-binding pocket in the catalytic core of PDK1 that was originally termed the PIF-binding pocket (6, 10).Besides its role in the docking of substrates to PDK1, the HM/PIF-binding pocket was also identified as a ubiquitous and key regulatory site in likely all AGC kinases (7, 11). Thus, in AGC kinases studied up to now, the HM/PIF-binding pocket serves as an intramolecular docking site for the phosphorylated HM. In summary, the HM has a dual function in AGC kinase activation, (i) mediating the intermolecular interaction with PDK1 and (ii) acting as an intramolecular allosteric activator that stabilizes the active conformation of the kinase domain via binding to the HM/PIF-binding pocket.The CT of AGC kinases additionally contains a second regulatory phosphorylation site traditionally termed the “turn motif” (TM), and more recently the zipper (Z) site. The Z/TM phosphate interacts with a binding site within the kinase domain, acting like a zipper which serves to support the intramolecular binding of the phosphorylated HM to the HM/PIF-binding pocket (12). Hence, AGC kinases are synergistically activated by phosphorylation at the activation loop, the HM, and the Z/TM sites.Protein kinase C-related protein kinases (PRKs) (13) (also named PAK for protease-activated kinase (14–16) and PKN for protein kinase N (17)) represent a subfamily of AGC kinases. So far, three PRK isoforms were identified, PRK1, PRK2, and PKN3, which are effectors of the small GTP-binding protein Rho. PRKs, as well as the Rho-associated kinases (ROCKs), are considered to be the protein kinases that mediate the phosphorylation events downstream of Rho activation and both can be inhibited by the highly specific protein kinase inhibitor Y27632 (18). The most notable role described for PRK2 is the control of entry into mitosis and exit from cytokinesis (19). In addition, PRK2 phosphorylates the hepatitis C virus (HCV) RNA polymerase (20). In support of a function in HCV RNA replication, PRK2 inhibitors like Y27632 suppress HCV replication (21).The N-terminal region of PRK2 possesses three Rho effector (HR1) domains (13), a pseudosubstrate region that is thought to have an autoinhibitory function (22) and a C2-like domain, which is a potential binding site for lipid activators. The C-terminal region of PRK2 harbors the HM that mediates the docking interaction with the HM/PIF-binding pocket in its upstream kinase PDK1 (10, 23). Interestingly, PRKs and also atypical protein kinase Cs (PKCs, PKCζ, and PKCι/λ), contain an acidic residue instead of a phosphorylatable amino acid at the site equivalent to the HM phosphorylation site in other AGC kinases. Therefore, the molecular events that regulate the interaction of PRK2 and PKCζ with PDK1 must be different from the mechanism characterized for S6K, SGK, and RSK.In the present work we extended and refined the model of docking interaction between PRK2 and PDK1 and characterized C-terminal regions of PRK2 that participate in the regulation of this interaction. The work sheds light on the common as well as specific mechanisms that operate in the regulation of PDK1 docking interaction with its different substrates.     

Interaction of the small G protein RhoA with the C terminus of human phospholipase D1

Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.     

Distribution of the small molecular weight GTP-binding proteins Rac1, Cdc42, RhoA and RhoB in the developing chick retina

Immunohistochemistry was used to determine the distribution of Rac1, Cdc42, RhoA and RhoB GTPases during development of the chick retina. All proteins appear as early as embryonic day 5 (E5) in cells of the vitreal margin, E7–8 in cells of the inner third of the inner nuclear layer and E9–10 in photoreceptors. From E10 until hatching, RhoA, Rac1 and Cdc42 were seen in perikarya and/or processes of amacrine, ganglion cells, and photoreceptors. Rho proteins were also observed in retinal Müller cells, with different distributions. RhoB showed a transient expression, being severely down regulated after E18. The distribution pattern of Rho proteins during the development of the chick retina suggests a concerted role in the differentiation of specific cell types, and probably during synaptogenesis.     

TGFbeta1-induced aortic endothelial morphogenesis requires signaling by small GTPases Rac1 and RhoA

TGFbeta is a potent regulator of cell differentiation in many cell types. On aortic endothelial cells, TGFbeta1 displays angiogenic properties in inducing capillary-like tube formation in collagen I gels, in vitro. We investigated cytoskeletal changes that precede tube formation and related these alterations to the effects of TGFbeta1 on the activation state of members of the RhoGTPase family. TGFbeta1 promotes cell elongation and stress fiber formation in aortic endothelial cells. Using cell lines with inducible expression of Rac1 mutants, we show that these events are mimicked by expression of dominant-negative Rac1 whereas the constitutively active mutant prevents the TGFbeta1-mediated change of phenotype. Although TGFbeta1 induces an initial rise in the Rac1-GTP content, this phase is followed by a prolonged loss of the active form. In contrast, RhoA activity increases progressively and reaches a plateau when Rac1-GTP is no longer detectable. Prolonged inhibition of Rac1 appears necessary and sufficient for the increase in RhoA-GTP. In situ examination of Rho activity in TGFbeta1-treated cells provides evidence that active RhoA relocalizes to the tips of elongated cells. Inhibiting the Rho effector ROCK abrogates tube formation. Thus, Rac1 and RhoA are regulated by TGFbeta1 in the process of endothelial tube formation in collagen I gels.     

Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase.

p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla. Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro. In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants. In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC). PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q). PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins. Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP. The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16). Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect. PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma. In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein. This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.     

Endocytosis of E-cadherin regulated by Rac and Cdc42 small G proteins through IQGAP1 and actin filaments

E-cadherin is a key cell-cell adhesion molecule at adherens junctions (AJs) and undergoes endocytosis when AJs are disrupted by the action of extracellular signals. To elucidate the mechanism of this endocytosis, we developed here a new cell-free assay system for this reaction using the AJ-enriched fraction from rat liver. We found here that non-trans-interacting, but not trans-interacting, E-cadherin underwent endocytosis in a clathrin-dependent manner. The endocytosis of trans-interacting E-cadherin was inhibited by Rac and Cdc42 small G proteins, which were activated by trans-interacting E-cadherin or trans-interacting nectins, which are known to induce the formation of AJs in cooperation with E-cadherin. This inhibition was mediated by reorganization of the actin cytoskeleton by Rac and Cdc42 through IQGAP1, an actin filament-binding protein and a downstream target of Rac and Cdc42. These results indicate the important role of the Rac/Cdc42-IQGAP1 system in the dynamic organization and maintenance of the E-cadherin-based AJs.     

Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1

Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.