Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis

Neutrophils exposed to chemoattractants polarize and accumulate polymerized actin at the leading edge. In neutrophil-like HL-60 cells, this asymmetry depends on a positive feedback loop in which accumulation of a membrane lipid, phosphatidylinositol (PI) 3,4,5-trisphosphate (PI[3,4,5]P3), leads to activation of Rac and/or Cdc42, and vice versa. We now report that Rac and Cdc42 play distinct roles in regulating this asymmetry. In the absence of chemoattractant, expression of constitutively active Rac stimulates accumulation at the plasma membrane of actin polymers and of GFP-tagged fluorescent probes for PI(3,4,5)P3 (the PH domain of Akt) and activated Rac (the p21-binding domain of p21-activated kinase). Dominant negative Rac inhibits chemoattractant-stimulated accumulation of actin polymers and membrane translocation of both fluorescent probes and attainment of morphologic polarity. Expression of constitutively active Cdc42 or of two different protein inhibitors of Cdc42 fails to mimic effects of the Rac mutants on actin or PI(3,4,5)P3. Instead, Cdc42 inhibitors prevent cells from maintaining a persistent leading edge and frequently induce formation of multiple, short lived leading edges containing actin polymers, PI(3,4,5)P3, and activated Rac. We conclude that Rac plays a dominant role in the PI(3,4,5)P3-dependent positive feedback loop required for forming a leading edge, whereas location and stability of the leading edge are regulated by Cdc42.     

Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase.

Ste20p from Saccharomyces cerevisiae belongs to the Ste20p/p65PAK family of protein kinases which are highly conserved from yeast to man and regulate conserved mitogen-activated protein kinase pathways. Ste20p fulfills multiple roles in pheromone signaling, morphological switching and vegetative growth and binds Cdc42p, a Rho-like small GTP binding protein required for polarized morphogenesis. We have analyzed the functional consequences of mutations that prevent binding of Cdc42p to Ste20p. The complete amino-terminal, non-catalytic half of Ste20p, including the conserved Cdc42p binding domain, was dispensable for heterotrimeric G-protein-mediated pheromone signaling. However, the Cdc42p binding domain was necessary for filamentous growth in response to nitrogen starvation and for an essential function that Ste20p shares with its isoform Cla4p during vegetative growth. Moreover, the Cdc42p binding domain was required for cell-cell adhesion during conjugation. Subcellular localization of wild-type and mutant Ste20p fused to green fluorescent protein showed that the Cdc42p binding domain is needed to direct localization of Ste20p to regions of polarized growth. These results suggest that Ste20p is regulated in different developmental pathways by different mechanisms which involve heterotrimeric and small GTP binding proteins.     

Regulation of phosphoinositide levels by the phospholipid transfer protein Sec14p controls Cdc42p/p21-activated kinase-mediated cell cycle progression at cytokinesis

Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade.     

Ras and the Rho Effector Cla4 Collaborate to Target and Anchor Lte1 at the Bud Cortex

Lte1, a protein important for exit from mitosis, localizes to the bud cortex as soon as the bud forms and remains there until cells exit from mitosis. Ras, the Rho GTPase Cdc42 and its effector the protein kinase Cla4 are required for Lte1’s association with the bud cortex. Here we investigate how Ras, and the Cdc42 effector Cla4 regulate the localization of Lte1. We find that Ras2 and Lte1 associate in stages of the cell cycle when Lte1 is phosphorylated and associated with the bud cortex and that this association requires CLA4. Additionally, RAS1 and RAS2 are required for CLA4-dependent Lte1 phosphorylation. Our findings suggest that Cla4-dependent phosphorylation promotes the initial association of Lte1 with Ras at the bud cortex and that Ras is required to stabilize phosphorylated forms of Lte1 at the bud cortex. Our results also raise the interesting possibility that the localization of Lte1 affects the protein’s ability to promote mitotic exit.     

Phosphorylation of the Cdc42 exchange factor Cdc24 by the PAK-like kinase Cla4 may regulate polarized growth in yeast

Rho-type GTPases control many cytoskeletal rearrangements, but their regulation remains poorly understood. Here, we show that in S. cerevisiae, activation of the CDK Cdc28-Cln2 at bud emergence triggers relocalization of Cdc24, the GEF for Cdc42, from the nucleus to the polarization site, where it is stably maintained by binding to the adaptor Bem1. Locally activated Cdc42 then polarizes the cytoskeleton in a manner dependent on its effectors Bni1 and the PAK-like kinase Cla4. In addition, Cla4 induces phosphorylation of Cdc24, leading to its dissociation from Bem1 at bud tips, thereby ending polarized bud growth in vivo. Our results thus suggest a dynamic temporal and spatial regulation of the Cdc42 module: Cdc28-Cln triggers actin polarization by activating Cdc42, which in turn restricts its own activation via a negative feedback loop acting on its GEF Cdc24.     

Control of Lte1 localization by cell polarity determinants and Cdc14

BACKGROUND: The putative guanine nucleotide exchange factor Lte1 plays an essential role in promoting exit from mitosis at low temperatures. Lte1 is thought to activate a Ras-like signaling cascade, the mitotic exit network (MEN). MEN promotes the release of the protein phosphatase Cdc14 from the nucleolus during anaphase, and this release is a prerequisite for exit from mitosis. Lte1 is present throughout the cell during G1 but is sequestered in the bud during S phase and mitosis by an unknown mechanism. RESULTS: We show that anchorage of Lte1 in the bud requires septins, the cell polarity determinants Cdc42 and Cla4, and Kel1. Lte1 physically associates with Kel1 and requires Kel1 for its localization in the bud, suggesting a role for Kel1 in anchoring Lte1 at the bud cortex. Our data further implicate the PAK-like protein kinase Cla4 in controlling Lte1 phosphorylation and localization. CLA4 is required for Lte1 phosphorylation and bud localization. Furthermore, when overexpressed, CLA4 induces Lte1 phosphorylation and localization to regions of polarized growth. Finally, we show that Cdc14, directly or indirectly, controls Lte1 dephosphorylation and delocalization from the bud during exit from mitosis. CONCLUSION: Restriction of Lte1 to the bud cortex depends on the cortical proteins Cdc42 and Kel1 and the septin ring. Cla4 and Cdc14 promote and demote Lte1 localization at and from the bud cortex, respectively, suggesting not only that the phosphorylation status of Lte1 controls its localization but also indicating that Cla4 and Cdc14 are key regulators of the spatial asymmetry of Lte1.     

Cla4 kinase triggers destruction of the Rac1-GEF Cdc24 during polarized growth in Ustilago maydis

Dimorphic switching from budding to filamentous growth is a characteristic feature of many pathogenic fungi. In the fungal model organism Ustilago maydis polarized growth is induced by the multiallelic b mating type locus and requires the Rho family GTPase Rac1. Here we show that mating type-induced polarized growth involves negative feedback regulation of the Rac1-specific guanine nucleotide exchange factor (GEF) Cdc24. Although Cdc24 is essential for polarized growth, its concentration is drastically diminished during filament formation. Cdc24 is part of a protein complex that also contains the scaffold protein Bem1 and the PAK kinase Cla4. Activation of Rac1 results in Cla4-dependent degradation of the Rac1-GEF Cdc24, thus creating a regulatory negative feedback loop. We generated mutants of Cdc24 that are resistant to Cla4-dependent destruction. Expression of stable Cdc24 variants interfered with filament formation, indicating that negative feedback regulation of Cdc24 is critical for the establishment of polarized growth.     

Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p

The p21-activated kinases (PAKs) are effectors for the Rho-family GTPase Cdc42p. Here we define the in vivo function of the kinase activity of the budding yeast PAK Cla4p, using cla4 alleles that are specifically inhibited by a cell-permeable compound that does not inhibit the wild-type kinase. CLA4 kinase inhibition in cells lacking the partially redundant PAK Ste20p causes reversible SWE1-dependent cell-cycle arrest and gives rise to narrow, highly elongated buds in which both actin and septin are tightly polarized to bud tips. Inhibition of Cla4p does not prevent polarization of F-actin, and cytokinesis is blocked only in cells that have not formed a bud before inhibitor treatment; cell polarization and bud emergence are not affected by Cla4p inhibition. Although localization of septin to bud necks is restored in swe1Delta cells, cytokinesis remains defective. Inhibition of Cla4p activity in swe1Delta cells causes a delay of bud emergence after cell polarization, indicating that this checkpoint may mediate an adaptive response that is capable of promoting budding when Cla4p function is reduced. Our data indicate that CLA4 PAK activity is required at an early stage of budding, after actin polarization and coincident with formation of the septin ring, for early bud morphogenesis and assembly of a cytokinesis site.     

Regulation of Gic2 localization and function by phosphatidylinositol 4,5-bisphosphate during the establishment of cell polarity in budding yeast

Establishment of cell polarity is important for a wide range of biological processes, from asymmetric cell growth in budding yeast to neurite formation in neurons. In the yeast Saccharomyces cerevisiae, the small GTPase Cdc42 controls polarized actin organization and exocytosis toward the bud. Gic2, a Cdc42 effector, is targeted to the bud tip and plays an important role in early bud formation. The GTP-bound Cdc42 interacts with Gic2 through the Cdc42/Rac interactive binding domain located at the N terminus of Gic2 and activates Gic2 during bud emergence. Here we identify a polybasic region in Gic2 adjacent to the Cdc42/Rac interactive binding domain that directly interacts with phosphatidylinositol 4,5-bisphosphate in the plasma membrane. We demonstrate that this interaction is necessary for the polarized localization of Gic2 to the bud tip and is important for the function of Gic2 in cell polarization. We propose that phosphatidylinositol 4,5-bisphosphate and Cdc42 act in concert to regulate polarized localization and function of Gic2 during polarized cell growth in the budding yeast.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast.

The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.     

Role of Cdc42-Cla4 interaction in the pheromone response of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.     

A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization

In Saccharomyces cerevisiae, activation of Cdc42 by its guanine-nucleotide exchange factor Cdc24 triggers polarization of the actin cytoskeleton at bud emergence and in response to mating pheromones. The adaptor protein Bem1 localizes to sites of polarized growth where it interacts with Cdc42, Cdc24 and the PAK-like kinase Cla4. We have isolated Bem1 mutants (Bem1-m), which are specifically defective for binding to Cdc24. The mutations map within the conserved PB1 domain, which is necessary and sufficient to interact with the octicos peptide repeat (OPR) motif of Cdc24. Although Bem1-m mutant proteins localize normally, bem1-m cells are unable to maintain Cdc24 at sites of polarized growth. As a consequence, they are defective for apical bud growth and the formation of mating projections. Localization of Bem1 to the incipient bud site requires activated Cdc42, and conversely, expression of Cdc42-GTP is sufficient to accumulate Bem1 at the plasma membrane. Thus, our results suggest that Bem1 functions in a positive feedback loop: local activation of Cdc24 produces Cdc42-GTP, which recruits Bem1. In turn, Bem1 stabilizes Cdc24 at the site of polarization, leading to apical growth.     

Phosphoinositides are essential coactivators for p21-activated kinase 1

Phospholipid-enriched membranes such as the plasma membrane can serve as direct regulators of kinase signaling. Pak1 is involved in growth factor signaling at the plasma membrane, and its dysregulation is implicated in cancer. Pak1 adopts an autoinhibited conformation that is relieved upon binding to membrane-bound Rho GTPases Rac1 or Cdc42, but whether lipids also regulate Pak1 in vivo is unknown. We show here that phosphoinositides, particularly PIP(2), potentiate Rho-GTPase-mediated Pak1 activity. A positively charged region of Pak1 binds to phosphoinositide-containing membranes, and this interaction is essential for membrane recruitment and activation of Pak1 in response to extracellular signals. Our results highlight an active role for lipids as allosteric regulators of Pak1 and suggest that Pak1 is a "coincidence detector" whose activation depends on GTPases present in phosphoinositide-rich membranes. These findings expand the role of phosphoinositides in kinase signaling and suggest how altered phosphoinositide metabolism may upregulate Pak1 activity in cancer cells.     

Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.     

Phosphoinositides specify polarity during epithelial organ development

The molecular mechanisms that integrate cellular polarity with tissue architecture during epithelial morphogenesis are poorly understood. Using a three-dimensional model of epithelial morphogenesis, report that the phosphatase PTEN and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] regulate the GTPase Cdc42 and the kinase aPKC to generate the apical plasma membrane domain and maintain apical-basolateral polarity.     

Two sphingolipid transfer proteins, CERT and FAPP2: their roles in sphingolipid metabolism

Recent discoveries of two sphingolipid transfer proteins, CERT and FAPP2, have brought the field of sphingolipid metabolism to a more dynamic stage. CERT transfers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, a step crucial for sphingomyelin (SM) synthesis. The pleckstrin homology (PH) domain and the FFAT motif of CERT restrict the direction of transfer and destination of ceramide through binding to phosphatidylinositol 4-monophosphate (PI4P) at the Golgi and the ER resident proteins, VAPs, respectively. CERT is regulated by the phosphorylation and dephosphorylation of serine/threonine, in which protein kinase D, possibly casein kinase I, and PP2Cepsilon are involved. On the other hand, FAPP2 transfers glucosylceramide (GlcCer) to appropriate sites for the synthesis of complex glycosphingolipids. Like CERT, FAPP2 contains a PH domain, the binding of which to PI4P is required for its localization to the Golgi. These observations indicate that lipid transfer proteins, CERT and FAPP2, spatially regulate lipid metabolism on the cytosolic side.     

Pleckstrin induces cytoskeletal reorganization via a Rac-dependent pathway.

Pleckstrin homology (PH) domains are present in over one hundred signaling molecules, where they are thought to mediate membrane targeting by binding to phosphoinositides. They were initially defined at the NH(2) and COOH termini of the molecule, pleckstrin, a major substrate for protein kinase C in platelets. We have previously reported that pleckstrin associates with the plasma membrane, where it induces the formation of villous and ruffled structures from the surface of transfected cells (1). We now show that overexpression of pleckstrin results in reorganization of the actin cytoskeleton. This pleckstrin effect is regulated by its phosphorylation and requires the NH(2)-terminal, but not the COOH-terminal, PH domain. Overexpression of the NH(2)-terminal PH domain alone of pleckstrin is sufficient to induce the cytoskeletal effects. Pleckstrin-induced actin rearrangements are not inhibited by pharmacologic inhibition of phosphatidylinositol 3-kinase, nor are they blocked by co-expression of a dominant negative phosphatidylinositol 3-kinase. The cytoskeletal effects of pleckstrin can be blocked by co-expression of a dominant negative Rac1 variant, but not wild-type Rac and not a dominant negative Cdc42 variant. These data indicate that the NH(2)-terminal PH domain of pleckstrin induces reorganization of the actin cytoskeleton via a pathway dependent on Rac but independent of Cdc42 and phosphatidylinositol 3-kinase.     

Essential role of the NH2-terminal region of Cdc24 guanine nucleotide exchange factor in its initial polarized localization in Saccharomyces cerevisiae

The cortical recruitment and accumulation of the small GTPase Cdc42 are crucial steps in the establishment of polarity, but this process remains obscure. Cdc24 is an upstream regulator of budding yeast Cdc42 that accelerates the exchange of GDP for GTP in Cdc42 via its Dbl homology (DH) domain. Here, we isolated five novel temperature-sensitive (ts) cdc24 mutants, the green fluorescent protein (GFP)-fused proteins of which lose their polarized localization at the nonpermissive temperature. All amino acid substitutions in the mutants were mapped to the NH2-terminal region of Cdc24, including the calponin homology (CH) domain. These Cdc24-ts mutant proteins did not interact with Bem1 at the COOH-terminal PB1 domain, suggesting a lack of exposure of the PB1 domain in the mutant proteins. The cdc24-ts mutants were also defective in polarization in the absence of Bem1. It was previously reported that a fusion protein containing Cdc24 and the p21-activated kinase (PAK)-like kinase Cla4 could bypass the requirement for Bem1 in polarity cue-independent budding (i.e., symmetry breaking). Cdc24-ts-Cla4 fusion proteins also showed ts localization at the polarity site. We propose that the NH2-terminal region unmasks the DH and PB1 domains, leading to the activation of Cdc42 and interaction with Bem1, respectively, to initiate cell polarization.     

Autophosphorylation of type I phosphatidylinositol phosphate kinase regulates its lipid kinase activity

Phosphatidylinositol phosphate kinases (PIPKs) have important roles in the production of various phosphoinositides. For type I PIP5Ks (PIP5KI), a broad substrate specificity is known. They phosphorylate phosphatidylinositol 4-phosphate most effectively but also phosphorylate phosphatidylinositol (PI), phosphatidylinositol 3-phosphate, and phosphatidylinositol (3,4)-bisphosphate (PI(3, 4)P(2)), resulting in the production of phosphatidylinositol (4, 5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol 3-phosphate, phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P(2)), phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P(2)), and phosphatidylinositol (3,4,5)-trisphosphate. We show here that PIP5KIs have also protein kinase activities. When each isozyme of PIP5KI (PIP5KIalpha, -beta, and -gamma) was subjected to in vitro kinase assay, autophosphorylation occurred. The lipid kinase-negative mutant of PIP5KIalpha (K138A) lost the protein kinase activity, suggesting the same catalytic mechanism for the lipid and the protein kinase activities. PIP5KIbeta expressed in Escherichia coli also retains this protein kinase activity, thus confirming that no co-immunoprecipitated protein kinase is involved. In addition, the autophosphorylation of PIP5KI is markedly enhanced by the addition of PI. No other phosphoinositides such as phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, or phosphatidylinositol trisphosphate have such an effect. We also found that the PI-dependent autophosphorylation strongly suppresses the lipid kinase activity of PIP5KI. The lipid kinase activity of PIP5KI was decreased to one-tenth upon PI-dependent autophosphorylation. All these results indicate that the lipid kinase activity of PIP5KI that acts predominantly for PI(4,5)P(2) synthesis is regulated by PI-dependent autophosphorylation in vivo.