Essential role of type I(alpha) phosphatidylinositol 4-phosphate 5-kinase in neurite remodeling

Rapid neurite remodeling is fundamental to nervous system development and plasticity and is regulated by Rho family GTPases that signal f-actin reorganization in response to various receptor ligands. Neuronal N1E-115 cells show dramatic neurite retraction and cell rounding in response to serum factors such as lysophosphatidic acid (LPA), sphingosine-1 phosphate (S1P), and thrombin, due to activation of the RhoA-Rho kinase pathway. Type I phosphatidylinositol 4-phosphate 5-kinases (PIPkinase), which regulate cellular levels of PtdIns(4,5)P(2), have been suggested as targets of the RhoA-Rho kinase pathway able to modulate cytoskeletal dynamics. Here, we show that the introduction of Type Ialpha PIPkinase into N1E-115 cells leads to cell rounding and complete inhibition of neurite outgrowth, perhaps through the dissociation of vinculin and the destabilization of focal adhesions. This occurs independently of RhoA, Rho kinase, and the activation of actomyosin contraction. Strikingly, expression of kinase-dead PIPkinase promotes the outgrowth of neurites, which fail to retract in response to LPA, S1P, thrombin, or active RhoA. Moreover, neurite retraction in response to an endogenous neuronal guidance cue, Semaphorin3A, was also dependent on Type Ialpha PIPkinase. Our results suggest an essential role for a Type I PIPkinase during neurite retraction in response to a number of diverse stimuli.     

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.     

Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.     

Diacylglycerol kinase zeta regulates phosphatidylinositol 4-phosphate 5-kinase Ialpha by a novel mechanism

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays an important role during actin polymerization and is produced by the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI), which are activated by phosphatidic acid (PA). As diacylglycerol kinases (DGKs) generate PA by phosphorylating diacylglycerol (DAG), we investigated whether DGKs were involved in controlling PIP2 levels by regulating PIP5KI activity. Here we show that expression of DGKzeta significantly enhances PIP5KIalpha activity in thrombin-stimulated HEK293 cells, and DGK activity is required for this stimulation. We also observed that DGKzeta co-immunoprecipitated and co-localized with PIP5KIalpha, suggesting that they reside in a regulated signaling complex. To explore the role of DGKzeta in actin polymerization, we examined the subcellular distribution of DGKzeta, PIP5KIalpha and actin, and found that these proteins co-localized with actin in lamellipodial protrusions. Supporting that PIP5KIalpha regulation occurs at the sites of actin polymerization, we found that PIP2 also accumulated in the actin-rich regions of lamellipodia. Significantly, in wounding assays, DGKzeta, PIP5KIalpha and PIP2 accumulated at the leading edge of migrating A172 cells, where massive actin polymerization is known to occur. Combined, these data support a novel function for DGKzeta: by generating PA, it stimulates PIP5KIalpha activity to increase local PIP2, which regulates actin polymerization.     

A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-ribosylation factor in Plasmodium falciparum

In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-ribosylation factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase β in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P₂ pathways allowing modulation of PtdIns(4,5)P₂ production in response to changes in intracellular calcium concentrations within the parasite.     

Phosphatidylinositol 4-phosphate 5-kinase type I is regulated through phosphorylation response by extracellular stimuli

Phosphatidylinositol 4-phosphate 5-kinase (PIPK) catalyzes a final step in the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a lipid signaling molecule. Strict regulation of PIPK activity is thought to be essential in intact cells. Here we show that type I enzymes of PIPK (PIPKI) are phosphorylated by cyclic AMP-dependent protein kinase (PKA), and phosphorylation of PIPKI suppresses its activity. Serine 214 was found to be a major phosphorylation site of PIPK type Ialpha (PIPKIalpha) that is catalyzed by PKA. In contrast, lysophosphatidic acid-induced protein kinase C activation increased PIPKIalpha activity. Activation of PIPKIalpha was induced by dephosphorylation, which was catalyzed by an okadaic acid-sensitive phosphatase, protein phosphatase 1 (PP1). In vitro dephosphorylation of PIPKIalpha with PP1 increased PIPK activity, indicating that PP1 plays a role in lysophosphatidic acid-induced dephosphorylation of PIPKIalpha. These results strongly suggest that activity of PIPKIalpha in NIH 3T3 cells is regulated by the reversible balance between PKA-dependent phosphorylation and PP1-dependent dephosphorylation.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

Regulation of Id2 gene expression by the insulin-like growth factor I receptor requires signaling by phosphatidylinositol 3-kinase

The Id proteins play an important role in proliferation, differentiation, and tumor development. We report here that Id gene expression can be regulated by the insulin-like growth factor I receptor (IGF-IR), a receptor that also participates in the regulation of cellular proliferation and differentiation. Specifically, we found that the IGF-IR activated by its ligand was a strong inducer of Id2 gene expression in 32D murine hemopoietic cells. This activation was not simply the result of cellular proliferation, as Id2 gene expression was higher in 32D cells stimulated by IGF-I than in cells exponentially growing in interleukin-3. The up-regulation of Id2 gene expression was largely dependent on the presence of insulin receptor substrate-1, a major substrate of the IGF-IR and a potent activator of the phosphatidylinositol 3-kinase (PI3K) pathway. The role of PI3K activity in the up-regulation of Id2 gene expression by the IGF-IR was confirmed by different methods and in different cell types. In 32D cells, the up-regulation of Id2 gene expression by the PI3K pathway correlated with interleukin-3 independence and inhibition of differentiation.     

Type I phosphatidylinositol 4-phosphate 5-kinase directly interacts with ADP-ribosylation factor 1 and is responsible for phosphatidylinositol 4,5-bisphosphate synthesis in the golgi compartment

Phosphatidylinositol (PtdIns) 4,5-bisphosphate is involved in many aspects of membrane traffic, but the regulation of its synthesis is only partially understood. Golgi membranes contain PI 4-kinase activity and a pool of phosphatidylinositol phosphate (PIP), which is further increased by ADP-ribosylation factor 1 (ARF1). COS7 cells were transfected with alpha and beta forms of PI 4-kinase, and only membranes from COS7 cells transfected with PI 4-kinase beta increased their content of PIP when incubated with ARF1. PtdIns(4, 5)P(2) content in Golgi membranes was nonexistent but could be increased to a small extent upon adding either cytosol or Type I or Type II PIP kinases. However, when ARF1 was present, PtdIns(4,5)P(2) levels increased dramatically when membranes were incubated in the presence of cytosol or Type I, but not Type II, PIP kinase. To examine whether ARF1 could directly activate Type I PIP 5-kinase, we used an in vitro assay consisting of phosphatidycholine-containing liposomes, ARF1, and PIP 5-kinase. ARF1 increased Type I PIP 5-kinase activity in a guanine nucleotide-dependent manner, identifying this enzyme as a direct effector for ARF1.     

Genomic tagging of endogenous type IIbeta phosphatidylinositol 5-phosphate 4-kinase in DT40 cells reveals a nuclear localisation

Previous studies from acutely transfected HeLa cells have identified an acidic alpha-helix in the Type IIbeta PtdIns5P 4-kinase (PIPkin IIbeta) as a putative novel nuclear localisation sequence (Ciruela et al. Biochem. J. 364, 587-591 2000). However, some heterogeneity in cellular localisation was always observed, and other published aspects of PIPkin IIbeta physiology are more consistent with an extra-nuclear function. As a means of resolving whether the endogenous PIPkin IIbeta is nuclear, we have used the high homologous recombination frequency of DT40 cells to knock an epitope tag (Mosedale et al., Nat Struct Biol. 12, 763-771 2005) into one of the alleles of the DT40 PIPkin IIbeta gene. We show that PIPkin IIbeta is expressed as a tagged protein, is active as revealed by immunoprecipitation and enzyme assay, and that cellular fractionation reveals that it is indeed nuclear. Genomic tagging of endogenous proteins in DT40 cells is a technique that offers unique advantages in studying endogenous signalling proteins.     

Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the rate-limiting step in the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a signaling phospholipid that contributes to actin dynamics. We have shown in transfected tissue culture cells that PIP5K translocates from the cytosol to the plasma membrane following agonist-induced stimulation of Rho family GTPases. Nonetheless, it is unclear whether Rho GTPases induce PIP5K relocalization in platelets. We used PIP5K isoform-specific immunoblotting and lipid kinase assays to examine the intracellular localization of PIP5K in resting and activated platelets. Using differential centrifugation to separate the membrane skeleton, actin filaments and associated proteins, and cytoplasmic fractions, we found that PIP5K isoforms were translocated from cytosol to actin-rich fractions following stimulation of the thrombin receptor. PIP5K translocation was detectable within 30 s of stimulation and was complete by 2-5 min. This agonist-induced relocalization and activation of PIP5K was inhibited by 8-(4-parachlorophenylthio)-cAMP, a cAMP analogue that inhibits Rho and Rac. In contrast, 8-(4-parachlorophenylthio)-cGMP, a cGMP analogue that inhibits Rac but not Rho, did not affect PIP5K translocation and activation. This suggests that Rho GTPase may be an essential regulator of PIP5K in platelets. Consistent with this hypothesis, we found that C3 exotoxin (a Rho-specific inhibitor) and HA1077 (an inhibitor of the Rho effector, Rho-kinase) also eliminated PIP5K activation and trafficking into the membrane cytoskeleton. Thus, these data indicate that Rho GTPase and its effector Rho-kinase have an intimate relationship with the trafficking and activation of platelet PIP5K. Moreover, these data suggest that relocalization of platelet PIP5K following agonist stimulation may play an important role in regulating the assembly of the platelet cytoskeleton.     

Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet alpha-granule secretion

To better understand the molecular mechanisms of platelet granule secretion, we have evaluated the role of type II phosphatidylinositol (PtdIns) 5-phosphate 4-kinase in agonist-induced platelet alpha-granule secretion. SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets was inhibited by either antibodies directed at type II PtdIns 5-phosphate 4-kinase or by a kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase. In contrast, recombinant type IIbeta PtdIns 5-phosphate 4-kinase augmented SFLLRN-stimulated alpha-granule secretion from SL-O-permeabilized platelets. SFLLRN-stimulated alpha-granule secretion was inhibited by a protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. Phorbol 12-myristate 13-acetate-stimulated alpha-granule secretion was inhibited by anti-type II PtdIns 5-phosphate 4-kinase antibodies or the kinase-impaired point mutant of type IIbeta PtdIns 5-phosphate 4-kinase and augmented by recombinant type IIbeta PtdIns 5-phosphate 4-kinase. Immunoblot analysis demonstrated that type II PtdIns 5-phosphate 4-kinase remained associated with SL-O-permeabilized platelets when incubated in the presence, but not the absence, of SFLLRN. This SFLLRN-induced translocation of type II PtdIns 5-phosphate 4-kinase was blocked by either the protein kinase C-specific inhibitor peptide or bisindolylmaleimide I. In addition to stimulating alpha-granule secretion, both SFLLRN and PMA enhanced the association of a fluorescein isothiocyanate-labeled peptide derived from the PtdIns (4,5)P(2)-binding domain of gelsolin to permeabilized platelets. Agonist-induced recruitment of the PtdIns (4,5)P(2)-binding domain was inhibited by neomycin, bisindolylmaleimide I, and anti-type II PtdIns 5-phosphate 4-kinase antibody. These results suggest a mechanism whereby protein kinase C-mediated translocation of type II PtdIns 5-phosphate 4-kinase leads to the recruitment of PtdIns (4,5)P(2)-binding proteins.     

Type Ialpha phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type Ialpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type Ialpha PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine alpha isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type Ialpha PIPkin. Under these conditions an increase in type Ialpha PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Ialpha PIPkin was co-transfected with either the human DGKepsilon isoform or a kinase-dead mutant of the murine DGKalpha isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Ialpha PIPkin by this lipid second messenger.     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.

We have found that insulin-like growth factor I (IGF-I) can protect fibroblasts from apoptosis induced by UV-B light. Antiapoptotic signalling by the IGF-I receptor depended on receptor kinase activity, as cells overexpressing kinase-defective receptor mutants could not be protected by IGF-I. Overexpression of a kinase-defective receptor which contained a mutation in the ATP binding loop functioned as a dominant negative and sensitized cells to apoptosis. The antiapoptotic capacity of the IGF-I receptor was not shared by other growth factors tested, including epidermal growth factor (EGF) and thrombin, although the cells expressed functional receptors for all the agonists. However, EGF was antiapoptotic for cells overexpressing the EGF receptor, and expression of activated pp60v-src also was protective. There was no correlation between protection from apoptosis and activation of mitogen-activated protein kinase, p38/HOG1, or p70S6 kinase. On the other hand, protection by any of the tyrosine kinases against UV-induced apoptosis was blocked by wortmannin, implying a role for phosphatidylinositol 3-kinase (PI3 kinase). To test this, we transiently expressed constitutively active or kinase-dead PI3 kinase and found that overexpression of activated phosphatidylinositol 3-kinase (PI3 kinase) was sufficient to provide protection against apoptosis. Because Akt/PKB is believed to be a downstream effector for PI3 kinase, we also examined the role of this serine/threonine protein kinase in antiapoptotic signalling. We found that membrane-targeted Akt was sufficient to protect against apoptosis but that kinase-dead Akt was not. We conclude that the endogenous IGF-I receptor has a specific antiapoptotic signalling capacity, that overexpression of other tyrosine kinases can allow them also to be antiapoptotic, and that activation of PI3 kinase and Akt is sufficient for antiapoptotic signalling.     

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.     

Intracellular movement of green fluorescent protein-tagged phosphatidylinositol 3-kinase in response to growth factor receptor signaling.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein-tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein-tagged fragments of p85 we show that binding to the receptor requires the NH(2)-terminal part of the protein as well as its SH2 domains.