Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.     

Engineering the phosphoinositide-binding profile of a class I pleckstrin homology domain

Pleckstrin homology (PH) domains are protein modules that bind with varying degrees of affinity and specificity membrane phosphoinositides. Previously we have shown that although the PH domains of the Ras GTPase-activating proteins GAP1m and GAP1IP4BP are 63% identical at the amino acid level they possess distinct phosphoinositide-binding profiles. The GAP1m PH domain binds phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas the domain from GAP1IP4BP binds PtdIns(3,4,5)P3 and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) equally well. These phosphoinositide specificities are translated into distinct subcellular localizations. GAP1m is cytosolic and undergoes a rapid PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. In contrast, GAP1IP4BP is constitutively associated, in a PtdIns(4,5)P2-dependent manner, with the plasma membrane (Cozier, G. E., Lockyer, P. J., Reynolds, J. S., Kupzig, S., Bottomley, J. R., Millard, T., Banting, G., and Cullen, P. J. (2000) J. Biol. Chem. 275, 28261-28268). In the present study, we have used molecular modeling to identify residues in the GAP1IP4BP PH domain predicted to be required for high affinity binding to PtdIns(4,5)P2. This has allowed the isolation of a mutant, GAP1IP4BP-(K591T), which while retaining high affinity for PtdIns(3,4,5)P3 has a 6-fold reduction in its affinity for PtdIns(4,5)P2. Importantly, GAP1IP4BP-(K591T) is predominantly localized to the cytosol and undergoes a PtdIns(3,4,5)P3-dependent association with the plasma membrane following growth factor stimulation. We have therefore engineered the phosphoinositide-binding profile of the GAP1IP4BP PH domain, thereby emphasizing that subtle changes in PH domain structure can have a pronounced effect on phosphoinositide binding and the subcellular localization of GAP1IP4BP.     

GAP1IP4BP contains a novel group I pleckstrin homology domain that directs constitutive plasma membrane association

The group I family of pleckstrin homology (PH) domains are characterized by their inherent ability to specifically bind phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and its corresponding inositol head-group inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P(4)). In vivo this interaction results in the regulated plasma membrane recruitment of cytosolic group I PH domain-containing proteins following agonist-stimulated PtdIns(3,4,5)P(3) production. Among group I PH domain-containing proteins, the Ras GTPase-activating protein GAP1(IP4BP) is unique in being constitutively associated with the plasma membrane. Here we show that, although the GAP1(IP4BP) PH domain interacts with PtdIns(3,4, 5)P(3), it also binds, with a comparable affinity, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) (K(d) values of 0.5 +/- 0.2 and 0.8 +/- 0.5 microm, respectively). Intriguingly, whereas this binding site overlaps with that for Ins(1,3,4,5)P(4), consistent with the constitutive plasma membrane association of GAP1(IP4BP) resulting from its PH domain-binding PtdIns(4,5)P(2), we show that in vivo depletion of PtdIns(4,5)P(2), but not PtdIns(3,4,5)P(3), results in dissociation of GAP1(IP4BP) from this membrane. Thus, the Ins(1,3,4,5)P(4)-binding PH domain from GAP1(IP4BP) defines a novel class of group I PH domains that constitutively targets the protein to the plasma membrane and may allow GAP1(IP4BP) to be regulated in vivo by Ins(1,3,4,5)P(4) rather than PtdIns(3,4,5)P(3).     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.     

Sprouty2 attenuates epidermal growth factor receptor ubiquitylation and endocytosis,and consequently enhances Ras/ERK signalling

Drosophila Sprouty (dSpry) was genetically identified as a novel antagonist of fibroblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGF- mediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DeltaN11 and mSpry4, both deficient in c-Cbl binding, to instigate these effects. hSpry2 interacts specifically with the c-Cbl RING finger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by specifically intercepting c-Cbl-mediated effects on receptor down-regulation.     

Differential roles of phosphatidylserine, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 in plasma membrane targeting of C2 domains. Molecular dynamics simulation, membrane binding, and cell translocation studies of the PKCalpha C2 domain

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca(2+)-dependent C2 domain of protein kinase Calpha (PKCalpha) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKCalpha-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), but that PS and Ca(2+) binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) augments the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca(2+)-dependent PS binding is essential for membrane interactions of PKCalpha-C2. PtdIns(4,5)P(2) alone cannot drive the membrane attachment of the domain but further stabilizes the Ca(2+)- and PS-dependent membrane binding. When the fluorescence protein-tagged PKCalpha-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P(2) and/or PtdIns(3,4,5)P(3) from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) at the PM slowed membrane dissociation of PKCalpha-C2. Collectively, these studies show that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) augment the Ca(2+)- and PS-dependent membrane binding of PKCalpha-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKCalpha-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.     

Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P(2). Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-D-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdIns(4,5)P(2), even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca(2+)/CaM binds with high affinity (K(d) approximately 10nM) to this domain and releases the PtdIns(4,5)P(2). MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P(2) (approx. 10 microM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P(2) buffer, binding PtdIns(4,5)P(2) in a quiescent cell, and releasing it locally when the intracellular Ca(2+) concentration increases. This reversible sequestration is important because PtdIns(4,5)P(2) plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.     

Phosphatidylinositol 4,5-bisphosphate is important for stomatal opening

Previously, we demonstrated that a protein that binds phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] inhibits both light-induced stomatal opening and ABA-induced stomatal closing. The latter effect is due to a reduction in free PtdIns(4,5)P(2), decreasing production of inositol 1,4,5-trisphosphate and phosphatidic acid by phospholipases C and D. However, it is less clear how PtdIns(4,5)P(2) modulates stomatal opening. We found that in response to white light irradiation, the PtdIns(4,5)P(2)-binding domain GFP:PLCdelta1PH translocated from the cytosol into the plasma membrane. This suggests that the level of PtdIns(4,5)P(2) increases at the plasma membrane upon illumination. Exogenously administered PtdIns(4,5)P(2) substituted for light stimuli, inducing stomatal opening and swelling of guard cell protoplasts. To identify PtdIns(4,5)P(2) targets we performed patch-clamp experiments, and found that anion channel activity was inhibited by PtdIns(4,5)P(2). Genetic analyses using an Arabidopsis PIP5K4 mutant further supported the role of PtdIns(4,5)P(2) in stomatal opening. The reduced stomatal opening movements exhibited by a mutant of Arabidopsis PIP5K4 (At3g56960) was countered by exogenous application of PtdIns(4,5)P(2). The phenotype of reduced stomatal opening in the pip5k4 mutant was recovered in lines complemented with the full-length PIP5K4. Together, these data suggest that PIP5K4 produces PtdIns(4,5)P(2) in irradiated guard cells, inhibiting anion channels to allow full stomatal opening.     

Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools

Regulation of PIPK (phosphatidylinositol phosphate kinase) and PtdIns(4,5)P2 signalling by small G-proteins and their effectors is key to many biological functions. Through selective recruitment and activation of different PIPK isoforms, small G-proteins such as Rho, Rac and Cdc42 modulate actin dynamics and cytoskeleton-dependent cellular events in response to extracellular signalling. These activities affect a number of processes, including endocytosis, bacterial penetration into host cells and cytolytic granule-mediated targeted cell killing. Small G-proteins and their modulators are also regulated by phosphoinositides through translocation and conformational changes. Arf family small G-proteins act at multiple sites as regulators of membrane trafficking and actin cytoskeletal remodelling, and regulate a feedback loop comprising phospholipase D, phosphatidic acid, PIPKs and PtdIns(4,5)P2, contributing to enhancement of PtdIns(4,5)P2-mediated cellular events and receptor signalling. Na+, Kir (inwardly rectifying K+), Ca2+ and TRP (transient receptor potential) ion channels are regulated by small G-proteins and membrane pools of PtdIns(4,5)P2. Yeast phosphatidylinositol 4-phosphate 5-kinases Mss4 and Its3 are involved in resistance against disturbance of sphingolipid biosynthesis and maintenance of cell integrity through the synthesis of PtdIns(4,5)P2 and downstream signalling through the Rom2/Rho2 and Rgf1/Rho pathways. Here, we review models for regulated intracellular targeting of PIPKs by small G-proteins and other modulators in response to extracellular signalling. We also describe the spatial and temporal cross-regulation of PIPKs and small G-proteins that is critical for a number of cellular functions.     

Structural and membrane binding analysis of the Phox homology domain of phosphoinositide 3-kinase-C2alpha

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of diverse PI specificities of PX domains, we determined the crystal structure of the PX domain from phosphoinositide 3-kinase C2alpha (PI3K-C2alpha), which binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). To delineate the mechanism by which this PX domain interacts with membranes, we measured the membrane binding of the wild type domain and mutants by surface plasmon resonance and monolayer techniques. This PX domain contains a signature PI-binding site that is optimized for PtdIns(4,5)P(2) binding. The membrane binding of the PX domain is initiated by nonspecific electrostatic interactions followed by the membrane penetration of hydrophobic residues. Membrane penetration is specifically enhanced by PtdIns(4,5)P(2). Furthermore, the PX domain displayed significantly higher PtdIns(4,5)P(2) membrane affinity and specificity when compared with the PI3K-C2alpha C2 domain, demonstrating that high affinity PtdIns(4,5)P(2) binding was facilitated by the PX domain in full-length PI3K-C2alpha. Together, these studies provide new structural insight into the diverse PI specificities of PX domains and elucidate the mechanism by which the PI3K-C2alpha PX domain interacts with PtdIns(4,5)P(2)-containing membranes and thereby mediates the membrane recruitment of PI3K-C2alpha.     

Tyrosine phosphorylation of Sprouty2 enhances its interaction with c-Cbl and is crucial for its function

Mammalian Sprouty (Spry) proteins are now established as receptor tyrosine kinase-induced modulators of the Ras/mitogen-activated protein kinase pathway. Specifically, hSpry2 inhibits the fibroblast growth factor receptor (FGFR)-induced mitogen-activated protein kinase pathway but conversely prolongs activity of the same pathway following epidermal growth factor (EGF) stimulation, where activated EGF receptors are retained on the cell surface. In this study it is demonstrated that hSpry2 is tyrosine-phosphorylated upon stimulation by either FGFR or EGF and subsequently binds endogenous c-Cbl with high affinity. A conserved motif on hSpry2, together with phosphorylation on tyrosine 55, is required for its enhanced interaction with the SH2-like domain of c-Cbl. A hSpry2 mutant (Y55F) that did not exhibit an enhanced binding with c-Cbl failed to retain EGF receptors on the cell surface. Furthermore, individually mutating hSpry2 residues 52-59 to alanine indicated a tight correlation between their affinity for c-Cbl binding and their inhibition of ERK2 activity in the FGFR pathway. We postulate that tyrosine phosphorylation "activates" hSpry2 by enhancing its interaction with c-Cbl and that this interaction is critical for its physiological function in a signal-specific context.     

Phosphatidylinositol-4,5-bisphosphate-rich plasma membrane patches organize active zones of endocytosis and ruffling in cultured adipocytes

A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cdelta1 (PLCdelta1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCdelta1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.     

Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells

Rapamycin (rapa)-induced heterodimerization of the FRB domain of the mammalian target of rapa and FKBP12 was used to translocate a phosphoinositide 5-phosphatase (5-ptase) enzyme to the plasma membrane (PM) to evoke rapid changes in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) levels. Rapa-induced PM recruitment of a truncated type IV 5-ptase containing only the 5-ptase domain fused to FKBP12 rapidly decreased PM PtdIns(4,5)P(2) as monitored by the PLCdelta1PH-GFP fusion construct. This decrease was paralleled by rapid termination of the ATP-induced Ca(2+) signal and the prompt inactivation of menthol-activated transient receptor potential melastatin 8 (TRPM8) channels. Depletion of PM PtdIns(4,5)P(2) was associated with a complete blockade of transferrin uptake and inhibition of epidermal growth factor internalization. None of these changes were observed upon rapa-induced translocation of an mRFP-FKBP12 fusion protein that was used as a control. These data demonstrate that rapid inducible depletion of PM PtdIns(4,5)P(2) is a powerful tool to study the multiple regulatory roles of this phospholipid and to study differential sensitivities of various processes to PtdIns(4,5)P(2) depletion.     

Coordinated intracellular translocation of phosphoinositide-specific phospholipase C-delta with the cell cycle

The delta family phosphoinositide (PI)-specific phospholipase C (PLC) are most fundamental forms of eukaryotic PI-PLCs. Despite the presence of lipid targeting domains such as the PH domain and C2 domain, the isoforms are also found in the cytoplasm and nucleus as well as at the plasma membrane. The isoforms have sequences or regions that can serve as a nuclear localization signal (NLS) and a nuclear export signal (NES). Their intracellular localization differs from one isoform to another, presumably due to the difference in the transport equilibrium balanced by the strength of the two signals of each isoform. Even for a particular isoform, its intracellular localization seems to vary during the cell cycle. As an example, PLCdelta(1), which is generally found at the plasma membrane and in the cytoplasm of quiescent cells, localizes to discrete nuclear structures in the G(1)/S boundary of the cell cycle. This may be at least partly due to an increase in intracellular Ca(2+), since Ca(2+) facilitates the formation of a nuclear transport complex comprised of PLCdelta(1) and importin beta1, a carrier molecule for the nuclear import. PLCdelta(1) as well as PLCdelta(4) may play a pivotal role in controlling the initiation of DNA synthesis in S phase. Spatio-temporal changes in the levels of PtdIns(4,5)P(2) seem to be another major determinant for the localization and regulation of the delta isoforms. High nuclear PtdIns(4,5)P(2) levels are associated with the G(1)/S phases. After entering M phase, PtdIns(4,5)P(2) synthesis at sites of cell division occurs and PLCs seem to localize to the cleavage furrow during cytokinesis. Coordinated translocation of PLCs with the cell cycle or with stress responses may result in changes in intra-nuclear environments and local membrane architectures that modulate proliferation and differentiation. In this review, recent findings regarding the molecular machineries and mechanisms of the nucleocytoplasmic shuttling as well as roles in the cell cycle progression of the delta isoforms of PLC will be discussed.     

The yeast inositol polyphosphate 5-phosphatases inp52p and inp53p translocate to actin patches following hyperosmotic stress: mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations

The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and PtdIns(3, 5)P(2). Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.     

Movin' on up: the role of PtdIns(4,5)P(2) in cell migration

Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.     

Identification of secretory granule phosphatidylinositol 4,5-bisphosphate-interacting proteins using an affinity pulldown strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.     

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway

The C2 domain is a targeting domain that responds to intracellular Ca²⁺ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P₂ specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca²⁺ specifically binds to the Ca²⁺-binding region, facilitating PtdIns(4,5)P₂ access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P₂ binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P₂ binding are essential for the plasma membrane localization of PKCα when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P₂ and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P₂ is hydrolyzed to generate diacylglycerol and IP₃, an important amount still remains in the membrane where it is available to activate PKCα. These findings entail revision of the currently accepted model of PKCα recruitment to the membrane and its activation.