Protein kinase C alpha phosphorylates and negatively regulates diacylglycerol kinase zeta

Diacylglycerol kinase (DGK) terminates diacylglycerol (DAG) signaling by phosphorylating DAG to produce phosphatidic acid, which also has signaling properties. Thus, precise control of DGK activity is essential for proper signal transduction. We demonstrated previously that a peptide corresponding to the myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation site domain (PSD) in DGK zeta was phosphorylated in vitro by an active fragment of protein kinase C (PKC). In the present study, we tested full-length DGK zeta and found that PKC alpha phosphorylated DGK zeta on serines within the MARCKS PSD in vitro and in vivo. DGK zeta also coimmunoprecipitated with PKC alpha, suggesting that they reside in a regulated signaling complex. We then tested whether phosphorylation affected DAG kinase activity. We found that a mutant (DGK zeta S/D) in which serines within the MARCKS PSD were altered to aspartates (to mimic phosphorylation) had lower activity compared with wild-type DGK zeta or a control mutant (DGK zeta S/N) in which the same serines were changed to asparagines. Furthermore, activation of PKC alpha by phorbol 12-myristate 13-acetate inhibited the activity of wild-type DGK zeta, but not DGK zeta S/D, in human embryonic kidney 293 cells. These results suggest that by phosphorylating the MARCKS PSD, PKC alpha attenuates DGK zeta activity. Supporting this, we found that cells expressing DGK zeta S/D had higher DAG levels and grew more rapidly compared with cells expressing DGK zeta S/N that could not be phosphorylated. Taken together, these results indicate that PKC alpha phosphorylates DGK zeta in cells, and this phosphorylation inhibits its kinase activity to remove cellular DAG, thereby affecting cell growth.     

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.     

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 zeta and eta in murine epidermis. TPA induces down-regulation of PKC eta but not PKC zeta.

Murine epidermis contains PKC zeta and eta as evidenced by the application of specific antisera. PKC zeta predominates in the cytosol and PKC eta in the particulate fraction. PKC zeta is shown to be present also in other murine tissues, with large amounts found in lung. Whereas epidermal PKC eta is completely down-regulated by treatment of mouse skin with TPA or bryostatin 1 for 18 h, PKC zeta is neither translocated by treatment with TPA for 20 min, nor down-regulated by treatment with TPA or bryostatin 1 for 18 h. PKC zeta is activated by phosphatidyl serine alone and does neither respond to Ca2+ nor to TPA. It is inhibited by staurosporine with an IC50 of 16 nM, which is within the same range of other PKC isoenzymes. The sensitivity of PKC zeta towards the staurosporine derivative K252a is similar to that of PKC alpha,beta,gamma but much higher than that of PKC delta and epsilon.     

Protein kinase C activation inhibits tyrosine phosphorylation of Cbl and its recruitment of Src homology 2 domain-containing proteins.

One of the major proteins that is rapidly tyrosine phosphorylated upon stimulation of the TCR/CD3 complex is the 120-kDa product of the c-cbl protooncogene (Cbl). Upon activation, tyrosine-phosphorylated Cbl interacts with the Src homology 2 (SH2) domains of several signaling proteins, e.g., phosphatidylinositol 3-kinase (PI3-K) and CrkL. In the present study, we report that pretreatment of Jurkat T cells with PMA reduced the anti-CD3-induced tyrosine phosphorylation of Cbl and, consequently, its activation-dependent association with PI3-K and CrkL. A specific protein kinase C (PKC) inhibitor (GF-109203X) reversed the effect of PMA on tyrosine phosphorylation of Cbl and restored the activation-dependent association of Cbl with PI3-K and CrkL. We also provide evidence that PKCalpha and PKCtheta can physically associate with Cbl and are able to phosphorylate it in vitro and in vivo. Furthermore, a serine-rich motif at the C terminus of Cbl, which is critical for PMA-induced 14-3-3 binding, is also phosphorylated by PKCalpha and PKCtheta in vitro. These results suggest that, by regulating tyrosine and serine phosphorylation of Cbl, PKC is able to control the association of Cbl with signaling intermediates, such as SH2 domain-containing proteins and 14-3-3 proteins, which may consequently result in the modulation of its function.     

Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.     

Protein kinase C regulates the phosphorylation and cellular localization of occludin

Occludin is an integral membrane phosphoprotein specifically associated with tight junctions, contributing to the structure and function of this intercellular seal. Occludin function is thought to be regulated by phosphorylation, but no information is available on the molecular pathways involved. In the present study, the involvement of the protein kinase C pathway in the regulation of the phosphorylation and cellular distribution of occludin has been investigated. Phorbol 12-myristate 13-acetate and 1,2-dioctanoylglycerol induced the rapid phosphorylation of occludin in Madin-Darby canine kidney cells cultured in low extracellular calcium medium with a concomitant translocation of occludin to the regions of cell-cell contact. The extent of occludin phosphorylation as well as its incorporation into tight junctions induced by protein kinase C activators or calcium switch were markedly decreased by the protein kinase C inhibitor GF-109203X. In addition, in vitro experiments showed that the recombinant COOH-terminal domain of murine occludin could be phosphorylated by purified protein kinase C. Ser(338) of occludin was identified as an in vitro protein kinase C phosphorylation site using peptide mass fingerprint analysis and electrospray ionization tandem mass spectroscopy. These findings indicate that protein kinase C is involved in the regulation of occludin function at tight junctions.     

Protein kinase C-mediated phosphorylation of retinal rod outer segment membrane proteins

We have previously reported that the purified GDP-bound alpha-subunit of the GTP-binding protein transducin (TD), present in outer segments of retinal rod cells (ROS), serves as a high affinity substrate (Km = 1 microM) for protein kinase C (PKC) [Zick et al. (1986) Proc. natn. Acad. Sci., U.S.A. 83, 9294-9297]. In the present study we demonstrate that TD-alpha undergoes phosphorylation by PKC when present in its native form in intact ROS membranes. This phosphorylation is inhibited by GTP-gamma-S which activates TD, suggesting that it is only the inactive conformation of TD-alpha that serves as a substrate for PKC. Indeed, both vanadate and AlF4, that confer an active conformation on TD-alpha-GDP, inhibit PKC-mediated phosphorylation of purified TD-alpha-GDP. We demonstrate that the purified beta subunit of TD also serves as an in vitro substrate for PKC. Moreover, following their phosphorylation, both TD-alpha and beta form high affinity complexes with PKC. This is evident from the findings that PKC coprecipitates with both the alpha and beta subunits of TD when the latter are immunoprecipitated by their respective antibodies. PKC phosphorylates additional ROS proteins of 36, 48 and 92 kDa, tentatively identified as rhodopsin, arrestin and the cGMP-phosphodiesterase. Taken together our results strongly suggest that phosphorylation of TD is of physiological relevance and that through phosphorylation of endogenous ROS proteins, PKC could play a key role in regulating phototransduction.     

hMutS alpha is protected from ubiquitin-proteasome-dependent degradation by atypical protein kinase C zeta phosphorylation

The hMutS alpha (hMSH2-hMSH6) protein heterodimer plays a critical role in the detection of DNA mispairs in the mismatch repair (MMR) process. We recently reported that hMutS alpha proteins were degraded by the ubiquitin-proteasome pathway in a cell-type-dependent manner, indicating that one or several regulator(s) may interfere with hMutS alpha protein ubiquitination and degradation. On the other hand, we and others have shown that protein kinase C (PKC) is involved as a positive regulator of MMR activity. Here, we provide evidence that the atypical PKC zeta regulates ubiquitination, degradation, and levels of hMutS alpha proteins. Using both PKC zeta-transfected U937 and PKC zeta siRNA-transfected MRC-5 cell lines, we found that PKC zeta protein expression was correlated with that of hMutS alpha as well as with MMR activity, but was inversely correlated with hMutS alpha protein ubiquitination and degradation. Interestingly, PKC zeta interacts with hMSH2 and hMSH6 proteins and phosphorylates both. Moreover, in an in vitro assay PKCzeta mediates phosphorylation events decreasing hMutS alpha protein degradation via the ubiquitin-proteasome pathway. Altogether, our results indicate that PKC zeta modulates hMutS alpha stability and protein levels, and suggest a role for PKC zeta in genome stability by regulating MMR activity.     

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase.

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.     

Protein kinase C catalyzes phosphorylation of guanylate cyclase in vitro

Protein kinase C catalyzes phosphorylation of purified rat brain guanylate cyclase. The phosphorylation is marked by concomitant increase in guanylate cyclase activity. TPA further enhances both phosphorylation and activity of guanylate cyclase. Data seem to provide clues to the molecular mechanism of one of the transformation-like responses mimicked by 12-O-tetradecanoylphorbol-13-acetate, i.e. the elevation of cyclic GMP. It is envisaged that protein kinase C may have a central role in the understanding of molecular events triggering carcinogenesis.     

Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy

Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.     

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.     

Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca2(+)-stimulated actomyosin MgATPase activity.

Effects of troponin phosphorylation on Ca2(+)-stimulated MgATPase activity of bovine cardiac actomyosin were examined. Phosphorylation by protein kinase C of troponin I and troponin T subunits in troponin or troponin-tropomyosin complex resulted in a decreased Ca2(+)-stimulated MgATPase activity in reconstituted actomyosin, and this effect was reversed by subsequent dephosphorylation by protein phosphatase 1. It was further observed that protein kinase C phosphorylation of either troponin I or troponin T subunits led to a similar inhibition of Ca2(+)-stimulated actomyosin MgATPase activity. In all cases, EC50 values (concentrations causing 50% stimulation) for Ca2+ were not appreciably affected by troponin phosphorylation by protein kinase C. Data from phosphorylation site analysis suggests that phosphorylation of threonine 144 in troponin I and possibly threonine 280 or threonine 199 in troponin T might be important for the observed decrease of Ca2(+)-stimulated actomyosin MgATPase. It is suggested that inhibition of actomyosin MgATPase caused by protein kinase C phosphorylation of troponin I and/or troponin T represents a new mechanism that can account for in part the reported negative inotropic effect of phorbol esters on various cardiac preparations.     

Characterization of the binding and phosphorylation of cardiac calsequestrin by epsilon protein kinase C.

In this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an epsilonprotein kinase C-unique sequence (epsilonV1) derived form the epsilonprotein kinase C regulatory domain. Calsequestrin binds activated epsilonprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between epsilonprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the epsilonprotein kinase C. Finally, we show that calsequestrin is an epsilonprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of epsilonprotein kinase C to calsequestrin.