The pleckstrin homology domain proteins Slm1 and Slm2 are required for actin cytoskeleton organization in yeast and bind phosphatidylinositol-4,5-bisphosphate and TORC2

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a key second messenger that regulates actin and membrane dynamics, as well as other cellular processes. Many of the effects of PtdIns(4,5)P(2) are mediated by binding to effector proteins that contain a pleckstrin homology (PH) domain. Here, we identify two novel effectors of PtdIns(4,5)P(2) in the budding yeast Saccharomyces cerevisiae: the PH domain containing protein Slm1 and its homolog Slm2. Slm1 and Slm2 serve redundant roles essential for cell growth and actin cytoskeleton polarization. Slm1 and Slm2 bind PtdIns(4,5)P(2) through their PH domains. In addition, Slm1 and Slm2 physically interact with Avo2 and Bit61, two components of the TORC2 signaling complex, which mediates Tor2 signaling to the actin cytoskeleton. Together, these interactions coordinately regulate Slm1 targeting to the plasma membrane. Our results thus identify two novel effectors of PtdIns(4,5)P(2) regulating cell growth and actin organization and suggest that Slm1 and Slm2 integrate inputs from the PtdIns(4,5)P(2) and TORC2 to modulate polarized actin assembly and growth.     

The yeast PH domain proteins Slm1 and Slm2 are targets of sphingolipid signaling during the response to heat stress

The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P(2), TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization.     

The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation

The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4(ts) mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.     

Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase

The essential phospholipid PI4,5P(2) is generated by a well conserved PI4P 5-kinase, Mss4, in yeast. Balanced production and turnover of PI4,5P(2) is important for normal organization of the actin cytoskeleton and cell viability. Previous studies have shown that multiple PI phosphatases can regulate PI4,5P(2) levels. We report a new, unexpected regulatory mechanism for PI4,5P(2) homeostasis, directed by nuclear-cytoplasmic shuttling of the lipid kinase. We show that Mss4 is a phosphoprotein, which contains a functional nuclear localization signal (NLS) and can shuttle between the cytoplasm and the nucleus. Temperature-conditional mss4 cells that accumulate Mss4 protein in the nucleus exhibit reduced levels of PI4,5P(2), depolarization of the actin cytoskeleton and a block in Mss4 phosphorylation, suggesting an essential role for phosphorylated Mss4 at the plasma membrane. Through the isolation of gene dosage-dependent suppressors of mss4 mutants, we identified Bcp1, a protein enriched in the nucleus, which is required for Mss4 nuclear export and is related to the mammalian BRCA2-interacting protein BCCIP. Together, these studies suggest a new mechanism for lipid kinase regulation through regulated nuclear-cytoplasmic shuttling.     

Stt4 PI 4-kinase localizes to the plasma membrane and functions in the Pkc1-mediated MAP kinase cascade

Production of the essential phospholipid PI4P at the Golgi by the Pik1 kinase is required for protein secretion, while a distinct pool of PI4P generated by the Stt4 kinase is critical for normal actin cytoskeleton organization. We identify a transmembrane protein that stabilizes Stt4 at the plasma membrane where it directs synthesis of PI4P, which is required for activation of the Rho1/Pkc1-mediated MAP kinase cascade. Inactivation of Stt4 or the PI4P 5-kinase Mss4 results in mislocalization of the Rho-GTPase GEF Rom2. Rom2 binds PI4,5P(2) through its PH domain and represents the first identified effector in the Stt4-Mss4 pathway. Based on these results, we propose that Stt4-Mss4 generates PI4,5P(2) at the plasma membrane, required to recruit/activate effector proteins such as Rom2.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Plasma membrane proteins Slm1 and Slm2 mediate activation of the AGC kinase Ypk1 by TORC2 and sphingolipids in S. cerevisiae

The PH domain-containing proteins Slm1 and Slm2 were originally identified as substrates of the rapamycin-insensitive TOR complex 2 (TORC2) and as mediators of signaling by the lipid second messenger phosphatidyl-inositol-4,5-bisphosphate (PI4,5P2) in budding yeast S. cerevisiae. More recently, these proteins have been identified as critical effectors that facilitate phosphorylation and activation of the AGC kinases Ypk1 and Ypk2 by TORC2.¹ Here, we review the molecular basis for this regulation as well as place it within the context of recent findings that have revealed Slm1/2 and TORC2-dependent phosphorylation of Ypk1 is coupled to the biosynthesis of complex sphingolipids and to their levels within the plasma membrane (PM) as well as other forms of PM stress. Together, these studies reveal the existence of an intricate homeostatic feedback mechanism, whereby the activity of these signaling components is linked to the biosynthesis of PM lipids according to cellular need.     

Regulation of podosomes by integrin alphavbeta3 and Rho GTPase-facilitated phosphoinositide signaling

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.     

Stimulation of phosphatidylinositol-4-phosphate 5-kinase by Rho-kinase

The serine/threonine kinase Rho-kinase was recently identified as a downstream effector of the small GTPase Rho, mediating effects of Rho on the actin cytoskeleton. Also phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) has been implicated in the regulation of actin polymerization. As the synthesis of PI(4,5)P(2) has been suggested to be affected by Rho proteins, we investigated whether Rho-kinase is involved in the control of PI(4,5)P(2) levels. Overexpression of RhoA in HEK-293 cells increased phosphatidylinositol 4-phosphate (PI4P) 5-kinase activity and concomitantly enhanced cellular PI(4,5)P(2) levels, whereas overexpression of the Rho-inactivating C3 transferase decreased both PI4P 5-kinase activity and PI(4,5)P(2) levels. These effects of RhoA could be mimicked by overexpression of wild-type Rho-kinase and of the constitutively active catalytic domain of Rho-kinase, Rho-kinase-CAT. In contrast, a kinase-deficient mutant of Rho-kinase had no effect on PI4P 5-kinase activity. Importantly, the increase in PI4P 5-kinase activity and PI(4,5)P(2) levels by wild-type Rho-kinase, but not by Rho-kinase-CAT, was completely prevented by coexpression of C3 transferase, indicating that the effect of Rho-kinase was under the control of endogenous Rho. In cell lysates, addition of recombinant RhoA and Rho-kinase-CAT stimulated PI4P 5-kinase activity. Finally, the increase in PI(4,5)P(2) levels induced by both Rho-kinase-CAT and RhoA was reversed by the Rho-kinase inhibitor HA-1077. Our data suggest that Rho-kinase is involved in the Rho-controlled synthesis of PI(4,5)P(2) by PI4P 5-kinase.     

Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease

The Ca2+/calmodulin-dependent phosphatase calcineurin promotes yeast survival during environmental stress. We identified Slm1 and Slm2 as calcineurin substrates required for sphingolipid-dependent processes. Slm1 and Slm2 bind to calcineurin via docking sites that are required for their dephosphorylation by calcineurin and are related to the PXIXIT motif identified in NFAT. In vivo, calcineurin mediates prolonged dephosphorylation of Slm1 and Slm2 during heat stress, and this response can be mimicked by exogenous addition of the sphingoid base phytosphingosine. Slm proteins also promote the growth of yeast cells in the presence of myriocin, an inhibitor of sphingolipid biosynthesis, and regulation of Slm proteins by calcineurin is required for their full activity under these conditions. During heat stress, sphingolipids signal turnover of the uracil permease, Fur4. In cells lacking Slm protein activity, stress-induced endocytosis of Fur4 is blocked, and Fur4 accumulates at the cell surface in a ubiquitinated form. Furthermore, cells expressing a version of Slm2 that cannot be dephosphorylated by calcineurin display an increased rate of Fur4 turnover during heat stress. Thus, calcineurin may modulate sphingolipid-dependent events through regulation of Slm1 and Slm2. These findings, in combination with previous work identifying Slm1 and Slm2 as targets of Mss4/phosphatidylinositol 4,5-bisphosphate and TORC2 signaling, suggest that Slm proteins integrate information from a variety of signaling pathways to coordinate the cellular response to heat stress.     

Eukaryotic elongation factor 1A2 cooperates with phosphatidylinositol-4 kinase III beta to stimulate production of filopodia through increased phosphatidylinositol-4,5 bisphosphate generation

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a transforming gene product that is highly expressed in human tumors of the ovary, lung, and breast. eEF1A2 also stimulates actin remodeling, and the expression of this factor is sufficient to induce the formation of filopodia, long cellular processes composed of bundles of parallel actin filaments. Here, we find that eEF1A2 stimulates formation of filopodia by increasing the cellular abundance of cytosolic and plasma membrane-bound phosphatidylinositol-4,5 bisphosphate [PI(4,5)P(2)]. We have previously reported that the eEF1A2 protein binds and activates phosphatidylinositol-4 kinase III beta (PI4KIIIbeta), and we find that production of eEF1A2-dependent PI(4,5)P(2) and generation of filopodia require PI4KIIIbeta. Furthermore, PI4KIIIbeta is itself capable of activating both the production of PI(4,5)P(2) and the creation of filopodia. We propose a model for extrusion of filopodia in which eEF1A2 activates PI4KIIIbeta, and activated PI4KIIIbeta stimulates production of PI(4,5)P(2) and filopodia by increasing PI4P abundance. Our work suggests an important role for both eEF1A2 and PI4KIIIbeta in the control of PI(4,5)P(2) signaling and actin remodeling.     

Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

Our understanding of sphingolipid metabolism and functions in the baker's yeast Saccharomyces cerevisiae has progressed substantially in the past 2 years. Yeast sphingolipids contain a C26-acyl moiety, all of the genes necessary to make these long-chain fatty acids have been identified, and a mechanism for how chain length is determined has been proposed. Advances in understanding how the de novo synthesis of ceramide and complex sphingolipids is regulated have been made, and they demonstrate that the Target Of Rapamycin Complex 2 (TORC2) controls ceramide synthase activity. Other work shows that TORC2 regulates the level of complex sphingolipids in a pathway using the Slm1 and Slm2 proteins to control the protein phosphatase calcineurin, which regulates the breakdown of complex sphingolipids. The activity of Slm1 and Slm2 has also been shown to be regulated during heat stress by phosphoinositides and TORC2, along with sphingoid long-chain bases and the Pkh1 and Pkh2 protein kinases, to control the actin cytoskeleton, the trafficking of nutrient transporters, and cell viability. Together, these results provide the first molecular insights into understanding previous genetic interaction data that indicated a connection between sphingolipids and the TORC2 and phosphoinositide signaling networks. This new knowledge provides a foundation for greatly advancing our understanding of sphingolipid biology in yeast.     

A novel phosphatidylinositol 4,5-bisphosphate-binding domain targeting the Phg2 kinase to the membrane in Dictyostelium cells

Phg2 is a ser/thr kinase involved in adhesion, motility, actin cytoskeleton dynamics, and phagocytosis in Dictyostelium cells. In a search for Phg2 domains required for its localization to the plasma membrane, we identified a new domain interacting with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 4-phosphate (PI(4)P) membrane phosphoinositides. Deletion of this domain prevented membrane recruitment of Phg2 and proper function of the protein in the phagocytic process. Moreover, the overexpression of this PI(4,5)P₂-binding domain specifically had a dominant-negative effect by inhibiting phagocytosis. Therefore, plasma membrane recruitment of Phg2 is essential for its function. The PI(4,5)P₂-binding domain fused to GFP (green fluorescent protein) (GFP-Nt-Phg2) was also used to monitor the dynamics of PI(4,5)P₂ during macropinocytosis and phagocytosis. GFP-Nt-Phg2 disappeared from macropinosomes immediately after their closure. During phagocytosis, PI(4,5)P₂ disappeared even before the sealing of phagosomes as it was already observed in mammalian cells. Together these results demonstrate that PI(4,5)P₂ metabolism regulates the dynamics and the function of Phg2.     

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P(2) hydrolysis or increased PI(4,5)P(2) generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P(2) dictates the remodeling of actin necessary for completion of phagocytosis.     

TOR complex 2–Ypk1 signaling regulates actin polarization via reactive oxygen species

The evolutionarily conserved mTOR complex 2 (mTORC2) signaling pathway is an important regulator of actin cytoskeletal architecture and, as such, is a candidate target for preventing cancer cell motility and invasion. Remarkably, the precise mechanism(s) by which mTORC2 regulates the actin cytoskeleton have remained elusive. Here we show that in budding yeast, TORC2 and its downstream kinase Ypk1 regulate actin polarization by controlling reactive oxygen species (ROS) accumulation. Specifically, we find that TORC2-Ypk1 regulates actin polarization both by vacuole-related ROS, controlled by the phospholipid flippase kinase Fpk1 and sphingolipids, and by mitochondria-mediated ROS, controlled by the PKA subunit Tpk3. In addition, we find that the protein kinase C (Pkc1)/MAPK cascade, a well-established regulator of actin, acts downstream of Ypk1 to regulate ROS, in part by promoting degradation of the oxidative stress responsive repressor, cyclin C. Furthermore, we show that Ypk1 regulates Pkc1 activity through proper localization of Rom2 at the plasma membrane, which is also dependent on Fpk1 and sphingolipids. Together these findings demonstrate important links between TORC2/Ypk1 signaling, Fpk1, sphingolipids, Pkc1, and ROS as regulators of actin and suggest that ROS may play an important role in mTORC2-dependent dysregulation of the actin cytoskeleton in cancer cells.     

Involvement of Saccharomyces cerevisiae Avo3p/Tsc11p in maintaining TOR complex 2 integrity and coupling to downstream signaling

Target-of-rapamycin proteins (TORs) are Ser/Thr kinases serving a central role in cell growth control. TORs function in two conserved multiprotein complexes, TOR complex 1 (TORC1) and TORC2; the mechanisms underlying their actions and regulation are not fully elucidated. Saccharomyces TORC2, containing Tor2p, Avo1p, Avo2p, Avo3p/Tsc11p, Bit61p, and Lst8p, regulates cell integrity and actin organization. Two classes of avo3 temperature-sensitive (avo3(ts)) mutants that we previously identified display cell integrity and actin defects, yet one is suppressed by AVO1 while the other is suppressed by AVO2 or SLM1, defining two TORC2 downstream signaling mechanisms, one mediated by Avo1p and the other by Avo2p/Slm1p. Employing these mutants, we explored Avo3p functions in TORC2 structure and signaling. By observing binary protein interactions using coimmunoprecipitation, we discovered that the composition of TORC2 and its recruitment of the downstream effectors Slm1p and Slm2p were differentially affected in different avo3(ts) mutants. These molecular defects can be corrected only by expressing AVO3, not by expressing suppressors, highlighting the role of Avo3p as a structural and signaling scaffold for TORC2. Phenotypic modifications of avo3(ts) mutants by deletion of individual Rho1p-GTPase-activating proteins indicate that two TORC2 downstream signaling branches converge on Rho1p activation. Our results also suggest that Avo2p/Slm1p-mediated signaling, but not Avo1p-mediated signaling, links to Rho1p activation specifically through the Rho1p-guanine nucleotide exchange factor Tus1p.     

Identification and characterization of a phosphoinositide phosphate kinase homolog

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) plays a central role in regulating the actin cytoskeleton as a substrate for phosphoinositide 3-kinase and phospholipase C as well as by binding directly to proteins that control the processes of actin monomer sequestration, filament severing, capping, nucleation, cross-linking, and bundling (Ma, L., Cantley, L. C., Janmey, P. A., and Kirschner, M. W. (1998) J. Cell Biol. 140, 1125-1136; Hinchliffe, K. (2000) Curr. Biol. 10, R104-R1051). Three related phosphatidylinositol 4-phosphate 5-kinases (PI(4)P 5-kinases) have been identified in mammalian cells (types Ialpha, Ibeta, and Igamma) and appear to play distinct roles in actin remodeling. Here we have identified a fourth member of this family by searching the human genome and EST data bases. This new protein, which we have designated phosphatidylinositol phosphate kinase homolog (PIPKH), is expressed at relatively high levels in brain and testis. Immunoprecipitates of PIPKH expressed in mammalian cells contain PI(4)P 5-kinase activity, but this activity is not affected by mutations in residues that inactivate other type I PI(4)P 5-kinases. We show that the PI(4)P 5-kinase activity in PIPKH immunoprecipitates can be explained by the ability of PIPKH to heterodimerize with other type I PI(4)P 5-kinases. Transfection of 293t cells with PIPKH resulted in >8-fold increase in total phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) without a significant net increase in total PI(4,5)P(2). When coexpressed with PIPKH, green fluorescent protein (GFP) fusion construct of the pleckstrin homology domain from Bruton's tyrosine kinase (GFP-BTK-PH) localized in intracellular vesicular structures, suggesting an unusual intracellular site of PI(3,4,5)P(3) production. Finally, expression of PIPKH induced the reorganization of actin from predominantly stress fibers to predominantly foci and comets similar to those observed previously in cells infected with the intracellular pathogen Listeria or transfected with recombinant PIPKIalpha. These results suggest that PIPKH acts as a scaffold to localize and regulate type I PI(4)P 5-kinases and the synthesis of PI(3,4,5)P(3).     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

The LIM protein Ajuba regulates phosphatidylinositol 4,5-bisphosphate levels in migrating cells through an interaction with and activation of PIPKI alpha

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIalpha), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba(-/-) mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIalpha in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba(-/-) MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIalpha while inhibiting the activity of PIPKIIbeta. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIalpha enzyme activity.