Ca2+ phospholipid-dependent and independent phosphorylation of synthetic peptide substrates by protein kinase C

Several synthetic peptides reproducing fragments of protamines have been used as model substrates for Ca2+/phospholipid-dependent protein kinase C, tested both in the absence of any effector (basal conditions) and upon activation by either Ca2+ and phosphatidylserine (or diacylglycerol) or limited proteolysis. Only the peptide Arg4-Tyr-Gly-Ser-Arg6-Tyr [Ga(52-65)] shares the unique property of protamines of being readily phosphorylated even under basal conditions. Optimal activity in the absence of effectors is observed with Tris/HCl buffer pH 7.5; Pipes and Hepes are less effective at pH 7.5, and at pH 6.5 basal phosphorylation is reduced. Under the best conditions for basal phosphorylation of Ga(52-65), its derivative with ornithine replaced for arginine and those corresponding to its C-terminal fragments Gly-Ser-Arg6-Tyr [Ga(57-65)] and Gly-Ser-Arg3 [Ga(57-61)], as well as the peptides Pro-Arg5-Ser2-Arg-Pro-Val-Arg [Th(1-12)], Arg4-Tyr-Arg2-Ser-Thr-Val-Ala [Th(13-23)] and Arg2-Leu-Ser2-Leu-Arg-Ala are not significantly affected though all of them, like histones, are more or less readily phosphorylated upon activation of protein kinase C by Ca2+/phosphatidylserine. The peptide Ser2-Arg-Pro-Val-Arg [Th(7-12)] however, corresponding to the C-terminal part of Th(1-12), is not phosphorylated even in the presence of activators. Limited proteolysis can roughly mimic the Ca2+/phosphatidylserine effect inducing however different extents of activation depending on the nature of the peptide substrates. Our results support the following two conclusions. Basal phosphorylation by protein kinase C in the absence of any effector requires peptide substrates whose target residue(s) are included between two extended arginyl blocks and is also dependent on pH and nature of the buffer. Peptides having extended clusters of either arginyl or ornithyl residues on the C-terminal side of serine are also readily phosphorylated, but they need activation of protein kinase by either Ca2+/phosphatidylserine or limited proteolysis. The same is true of peptides having basic residues only on the N-terminal side, or even on both sides but in limited number.     

eNOS translocation but not eNOS phosphorylation is dependent on intracellular Ca2+ in human atrial myocardium

In endothelial cells, two ways of endothelial nitric oxide (NO) synthase (eNOS) activation are known: 1) translocation and 2) Akt-dependent phosphorylation of the enzyme at Ser¹¹⁷⁷ (Ser¹¹⁷⁷ eNOS). We have recently shown that agonist-induced Ser¹¹⁷⁷ eNOS phosphorylation also occurs in human myocardium (10). In this study, we investigated the Ca²⁺ dependency of these two mechanisms in human atrium. Therefore, atrial tissue was obtained from patients who underwent coronary artery bypass operations. In immunohistochemical experiments, the translocated form of eNOS and phosphorylated Ser¹¹⁷⁷ eNOS were labeled using specific antibodies. eNOS translocation was measured in the absence and presence of the Ca²⁺ chelator BAPTA before and after application of BRL 37344 (BRL), a ₃-adrenoceptor agonist that increases eNOS activity (34). In the absence of BAPTA, BRL time dependently increased the staining intensity of translocated eNOS, whereas in the presence of BAPTA, this effect was blunted. In contrast, BRL clearly increased the staining of phosphorylated Ser¹¹⁷⁷ eNOS even in the presence of BAPTA. This observation was confirmed using Western blot analysis. Using the NO-sensitive dye diaminofluorescein, we have demonstrated that BRL induced a strong NO release. This effect was completely abolished in the presence of BAPTA but was unaffected by LY-292004, an inhibitor of phosphatidylinositol 3-kinase activity and eNOS phosphorylation. Although Ca²⁺ dependent, neither the translocation of eNOS nor NO release was changed by the adenylate cyclase activator forskolin. In conclusion, 1) in human atrial myocardium, BRL-induced eNOS translocation but not Ser¹¹⁷⁷ eNOS phosphorylation is dependent on intracellular Ca²⁺. 2) In atrial myocardium, eNOS-translocation and not Ser¹¹⁷⁷ eNOS phosphorylation is responsible for generating the main amount of NO. 3) Although Ca²⁺ dependent, eNOS translocation and NO release could not be mimicked by adenylate cyclase activation as a mediator of -adrenergic stimulation. ₃-adrenoceptor; BRL 37344; cardiomyocyte; heart; Ca²⁺ regulation     

Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.     

A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.     

Insulin-stimulated activation of eNOS is independent of Ca2+ but requires phosphorylation by Akt at Ser(1179)

Vasodilator actions of insulin are mediated by activation of endothelial nitric-oxide synthase (eNOS) and subsequent production of NO. Phosphatidylinositol 3-kinase and Akt play important roles in insulin-signaling pathways leading to production of NO in vascular endothelium. Here we dissected mechanisms whereby insulin activates eNOS by using the fluorescent dye DAF-2 to directly measure NO production in single cells. Insulin caused a rapid increase in intracellular NO in NIH-3T3(IR) cells transiently transfected with eNOS. The stimulation of NO production by lysophosphatidic acid (LPA) was abrogated by pretreatment of cells with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Remarkably, in the same cells, insulin-stimulated production of NO was unaffected. However, cells expressing the eNOS-S1179A mutant (disrupted Akt phosphorylation site) did not produce detectable NO in response to insulin, whereas the response to LPA was similar to that observed in cells expressing wild-type eNOS. Moreover, production of NO in response to insulin was blocked by coexpression of an inhibitory mutant of Akt, whereas the response to LPA was unaffected. Phosphorylation of eNOS at Ser(1179) was observed only in response to treatment with insulin, but not with LPA. Interestingly, platelet-derived growth factor treatment of cells activated Akt but not eNOS. Results from human vascular endothelial cells were qualitatively similar to those obtained in transfected NIH-3T3(IR) cells, although the magnitude of the responses was smaller. We conclude that insulin regulates eNOS activity using a Ca(2+)-independent mechanism requiring phosphorylation of eNOS by Akt. Importantly, phosphorylation-dependent mechanisms that enhance eNOS activity can operate independently from Ca(2+)-dependent mechanisms.     

Protein kinase C inhibits Ca2+ accumulation in cardiac sarcoplasmic reticulum

It is now recognized that phorbol esters are negative inotropic agents in mammalian heart which presumably act via stimulation of Ca2(+)-activated phospholipid-dependent protein kinase (PKC). The goal in the present study was to identify the underlying cellular processes. Digitonin-permeabilized cultured neonatal rat ventricular myocytes were used to study biochemical and functional effects of phorbol esters on cardiac sarcoplasmic reticulum (SR). These cells contracted spontaneously at 3 microM Ca2+. Beating was inhibited by 10 microM ryanodine and was insensitive to 1 microM nifedipine. Thus, beating behavior results from the phasic oscillation of Ca2+ transport by SR in this preparation. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreased frequency by 30%, suggesting that Ca2+ transport by SR had been reduced. Whereas cAMP stimulated the rate of oxalate-supported 45Ca2+ uptake 2-fold, phorbol esters, TPA, and phorbol 12,13-dibutyrate inhibited this process by about 45%. The effects of phorbols were specific: (a) the alpha-analogues of TPA and phorbol 12,13-dibutyrate were inactive; and (b) the phorbol esters had no effect on Ca2+ transport in cells that had been depleted of PKC. TPA decreased oxalate-stimulated Ca2+ uptake over the entire range of Ca2+ concentrations, from 0.1 to 10 microM, by at least 70% without shifting the half-maximal effective Ca2+ concentration. Taken together these results indicate that the effects of phorbol ester on cardiac contraction are due to decreased Ca2+ transport by the SR and that these responses are mediated by PKC. These studies support the interpretation that the negative inotropic effects of phorbol esters are due, in part, to decreased SR function.     

Protein kinase a dependent phosphorylation activates mg2+ efflux in the basolateral region of the liver

Isolated hepatocytes in physiological [Na⁺] ₀ tightly maintain [Mg²⁺] _i . Upon β-adrenergic stimulation or in the presence of permeable cAMP, hepatocytes release 5–10% (1–3 mM Mg²⁺) of their total Mg²⁺ content. However, isolated basolateral liver plasma membranes (bLPM), release Mg²⁺ in the presence of [Na⁺] _o even in the absence of catecholamine stimulation. The data indicate that a physiological brake for Mg²⁺ efflux is present in the hepatocyte and is removed upon cellular signaling. In contrast, this regulation “brake” is absent in purified bLPM thus rendering them fully active. The present study was carried out to reconstruct the missing regulatory component. Activation of Mg²⁺ extrusion in intact cells is consistent with cAMP dependent phosphorylation of the transporter or a regulatory protein. Treatment of bLPM with a non-specific phosphatase such as alkaline phosphatase (AP), decreased Mg²⁺ efflux by 70% compared to untreated bLPM. When AP-treated bLPM were loaded with protein kinase A (PKA), and stimulated with permeable cAMP, Mg²⁺ transport fully recovered. These data suggest that phosphorylation of the Na⁺/Mg²⁺ exchanger or a nearby protein activates the Mg²⁺ transport mechanism in hepatocytes.     

Ca2+-phospholipid dependent phosphorylation of smooth muscle myosin

Isolated myosin light chain from chicken gizzard has been shown to serve as a substrate for Ca²⁺-activated phospholipid-dependent protein kinase. Autoradiography showed that Ca²⁺-activated phospholipid-dependent protein kinase phosphorylated mainly the 20,000-dalton light chain of chicken gizzard myosin. Exogenously added calmodulin had no effect on myosin light chain phosphorylation catalyzed by the enzyme. The 20,000-dalton myosin light chain, both in the isolated form and in the whole myosin form, served as the substrate for this enzyme. In contrast to the isolated myosin light chain, the light chain of whole myosin was phosphorylated to a lesser extent by the Ca²⁺-activated phospholipid dependent kinase. Our results suggest the involvement of phospholipid in regulating Ca²⁺-dependent phosphorylation of the 20,000-dalton light chain of smooth muscle myosin.     

Thrombin-stimulated Pyk2 phosphorylation in human endothelium is dependent on intracellular calcium and independent of protein kinase C and Src kinases

G-protein-coupled receptor agonists (GPCAs) cause functional responses in endothelial cells including secretion, proliferation, and altering monolayer permeability. These events are mediated in part by activation of the p42/44 mitogen-activated protein kinase (MAPK) cascade. The cytosolic tyrosine kinase Pyk2 is postulated to link GPCA-induced changes in intracellular calcium to activation of the MAP kinase cascade. We have investigated the regulation of Pyk2 in human umbilical vein endothelial cells in response to GPCAs and show that (1) thrombin, a PAR-1 peptide, and histamine cause rapid concentration- and time-dependent phosphorylation on tyrosines 402 (Src kinase binding site), 881 (Grb2 binding site), and 580 (an autophosphorylation site), (2) thrombin-stimulated phosphorylation is dependent on intracellular calcium and independent of PKC and PI-3 kinase, and (3) inhibition of Src kinases has no significant effect on thrombin-stimulated phosphorylation, implying that tyrosine phosphorylation of Pyk2 is independent of Src binding.     

Substitution of a single residue, Asp575, renders the NCKX2 K+-dependent Na+/Ca2+ exchanger independent of K+

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na(+) gradient and the outward K(+) gradient to drive Ca(2+) extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K(+) dependence of NCKX-mediated Ca(2+) transport. Charge-removing replacement of Asp(575) by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K(+), whereas the charge-conservative substitution of Asp(575) to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp(575) is conserved in the NCKX1-5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K(+)-independent Na(+)/Ca(2+) exchanger.     

Ca2+-activated, phospholipid-dependent protein kinase catalyzes the phosphorylation of actin-binding proteins

Chicken gizzard vinculin and filamin were found to be phosphorylated by Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). These two actin-binding proteins serve as substrates for protein kinase C specifically in the free form, whereas they are little phosphorylated by protein kinase C in the presence of F-actin. In contrast, alpha-actinin from chicken gizzard is less susceptible to phosphorylation by protein kinase C, either in the presence or in the absence of F-actin. In light of these data, the possibility that Ca2+ and phospholipid-dependent phosphorylation by protein kinase C may modulate the function of actin-binding proteins has to be considered.     

LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding

Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.     

Neuronal galvanotropism is independent of external Ca2+ entry or internal Ca2+ gradients

The mechanism by which growing neurites sense and respond to small applied electrical fields is not known, but there is some evidence that the entry of Ca²⁺ from the external medium, with the subsequent formation of intracellular Ca²⁺ gradients, is important in this process. We have employed two approaches to test this idea. Xenopus spinal neurites were exposed to electrical fields in a culture medium in which Ca²⁺ was chelated to very low levels compared to the normal extracellular concentration of 2 mM. In other experiments, loading the neurites with the calcium buffer, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA), disrupted the putative internal Ca²⁺ gradients, and the effects on the electrical response were determined. Fields of 100 mV/mm were applied for 12 h, and no difference was detected in the cathodal turning response between the treated neurites and the untreated controls. Using the Differential Growth Index (DGI), an asymmetry index, to quantitate the turning response, we recorded DGIs of −0.64, −0.65, and −0.62 for control cells, cells in Ca²⁺‐free medium, and cells preloaded with BAPTA, respectively. Furthermore, we detected an increase in neurite length for those neurons cultured in Ca²⁺‐free medium; they were 1.5–1.7 times as long as neurites from neurons cultured in normal Ca²⁺ medium. Likewise, we found that BAPTA‐loaded neurites were longer than control neurites. Our data indicate that neuronal galvanotropism is independent of the entry of external Ca²⁺ or of internal Ca²⁺ gradients. Both cell‐permeant agonistic and antagonistic analogs of cyclic 3′,5′‐adenosine monophosphate (cAMP) increased the response to applied electrical fields. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 30–38, 2000     

Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids

Protein kinase C has been shown to be a phospholipid/Ca2+-dependent enzyme activated by diacylglycerol (Nishizuka, Y. (1984) Nature 308, 693-697; Nishizuka, Y. (1984) Science 225, 1365-1370). We have reported that unsaturated fatty acids (oleic acid and arachidonic acid) can activate protein kinase C independently of Ca2+ and phospholipid (Murakami, K., and Routtenberg, A. (1985) FEBS Lett. 192, 189-193). This study shows that other cis-fatty acids such as linoleic acid also fully activate protein kinase C in the same manner. None of the saturated fatty acids (C:4 to C:18) nor the detergents (sodium dodecyl sulfate and Triton X-100) tested here were as effective as oleic acid. Unlike oleic acid, these detergents strongly inhibited protein kinase C activity induced by Ca2+/phosphatidylserine (PS) and diacylglycerol. Lowering the critical micelle concentration of oleic acid by increasing ionic strength also strongly inhibited oleic acid activation of protein kinase C activity. Dioleoylphosphatidylserine activated protein kinase C effectively (Ka = 7.2 microM). On the other hand, dimyristoylphosphatidylserine, which contains saturated fatty acids at both acyl positions, failed to activate protein kinase C even in the presence of Ca2+. These observations suggest that: protein kinase C activation by free fatty acid is specific to the cis-form and is not due to their detergent-like action, cis-fatty acid activation is due to the direct interaction of protein kinase C with the monomeric form of cis-fatty acids and not with the micelles of fatty acids, and cis-fatty acids at acyl positions in PS are also important for Ca2+/PS activation of protein kinase C.     

Multifunctional Ca2+/calmodulin-dependent protein kinase made Ca2+ independent for functional studies

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) that is transiently expressed in COS-7 cells is essentially inactive when assayed without Ca2+. Physiological activation of the kinase occurs by binding of Ca2+/calmodulin near a putative autoinhibitory subdomain that contains the sequence His282-Arg-Gln-Glu-Thr286. We have markedly increased the Ca2(+)-independent activity of CaM kinase by altering the charge of this sequence by site-directed mutagenesis. The mutant containing Asp282-Gly-Glu-Glu-Thr286 is 67% Ca2+ independent. We also mimicked the effect of autophosphorylation at Thr286 by the mutant containing His282-Arg-Gln-Glu-Asp286, which is 36% Ca2+ independent. In addition to delineating the autoinhibitory domain by use of mutations that disable it, these constructs are of immediate practical value for simulating CaM kinase action in vivo without elevating Ca2+. To this end, we show that nuclear microinjection of cDNA of a constitutive mutant, but not of the wild-type kinase, initiates maturation of Xenopus oocytes.     

Protein kinase C mediated activation and phosphorylation of Ca(2+)-pump in cardiac sarcolemma.

The effects of purified protein kinase C (PKC) on the Ca(2+)-pumping ATPase of cardiac sarcolemma were investigated. The addition of PKC to sarcolemmal vesicles resulted in a significant increase in ATP-dependent Ca2+ uptake, by increasing the calcium affinity by 2.8-fold (Km 0.14 vs. 0.4 microM for control) and by increasing Vmax from 5 to 6.8 nmol.mg protein-1.min-1. The addition of PKC also stimulated Ca2+ ATPase activity in sarcolemmal preparations. This activity was increased further upon the addition of calmodulin. These results suggest that PKC stimulates Ca2+ ATPase through a kinase-directed phosphorylation. The addition of PKC to a purified preparation of Ca2+ ATPase in the presence of [gamma-32P]ATP resulted in a 100% increase in phosphorylation that was dependent on the presence of Ca2+, phosphatidylserine, and phorbol 12,13-dibutyrate. These results demonstrate that the Ca2+ ATPase of canine cardiac muscle can be phosphorylated by PKC in vitro, resulting in increased affinity of the Ca2+ ATPase for Ca2+ and increase in the Ca2+ pump pumping rate. The results suggest that the Ca(2+)-pumping ATPase in heart tissue can be stimulated by PKC, thereby regulating the intracellular Ca2+ levels in whole heart.     

Protein kinase C isozyme-specific phosphorylation of profilin

Profilin, a cytoskeletal protein, is emerging as an important link between signal transduction pathways and cytoskeletal dynamics. Profilin is phosphorylated on its C-terminal serine by protein kinase C (PKC). The protein kinase used for the in vitro phosphorylation studies reported earlier was a mixture of isozymes, and therefore, attempts were made to address the isozyme specificity on profilin phosphorylation under in vitro conditions. Profilin was subjected to phosphorylation by PKCalpha, PKCepsilon, and PKCzeta isozymes individually, and it was observed that profilin phosphorylation is cofactor-independent. PKCzeta phosphorylates profilin to a higher extent, but exhibits cofactor dependency with respect to phosphoinositides. The stoichiometry of phosphorylation was measured in the presence of these different isozymes, and a maximum stoichiometry of 0.8 (mole phosphate incorporated/mole profilin) was obtained in the presence of PKCzeta. Phosphorylation of profilin by PKCzeta was maximal in the presence of phosphatidylinositol4,5-bisphosphate (PI4,5-P2) when compared to the other phosphoinositides studied.