Regulation of phospholipase D2 activity by protein kinase C alpha

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, G? 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.     

Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane

Activities of phospholipase D (PLD) in diverse subcellular organelles have been identified but the details of regulatory mechanisms in such locations are unknown. Protein kinase C (PKC) is a major regulator of PLD. Serine 2, threonine 147, and serine 561 residues of phospholipase D1 (PLD1) were determined as sites of phosphorylation by PKC (Kim, Y., Han, J. M., Park, J. B., Lee, S. D., Oh, Y. S., Chung, C., Lee, T. G., Kim, J. H., Park, S. K., Yoo, J. S., Suh, P. G., Ryu, S. H. (1999) Biochemistry 38, 10344-10351). In our present study, a triple mutation of these phosphorylation sites diminished markedly phorbol 12-myristate 13-acetate (PMA)-induced PLD1 activity in COS-7 cells. We looked at the location of the PLD1 phosphorylation by PKC by observing PMA induced band shifts and by use of anti-phospho-PLD1 monoclonal antibody. The shifted PMA-induced proteins and the immunoreactivity of the anti-phospho-PLD1 antibody were mainly found in the caveolin-enriched membrane (CEM) fraction. Depletion of cellular cholesterol led to a loss of this compartmentalization of phosphorylated PLD1 in the CEM. Replacement of the cellular cholesterol led to the restoration of phosphorylated PLD1 in the CEM. Immunocytochemical studies of COS-7 cells revealed that PLD1 was localized in the plasma membrane as well as in the vesicular structures in the cytoplasm, but the phosphorylation of PLD1 occurred only in the plasma membrane. Our results, therefore, show that phosphorylation, and thereby activation, of PLD1 by PKC occurs in the caveolin and cholesterol-enriched low density domain of the plasma membrane in COS-7 cells.     

Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells.

Many studies have shown that protein kinase C (PKC) is an important physiological regulator of phospholipase D (PLD). However, the role of PKC in agonist-induced PLD activation has been mainly investigated with a focus on the PLD1, which is one of the two PLD isoenzymes (PLD1 and PLD2) cloned to date. Since the expression of PLD2 significantly enhanced phorbol 12-myristate 13-acetate (PMA)- or bradykinin-induced PLD activity in rat pheochromocytoma PC12 cells, we investigated the regulatory mechanism of PLD2 in PC12 cells. Two different PKC inhibitors, GF109203X and Ro-31-8220, completely blocked PMA-induced PLD2 activation. In addition, specific inhibition of PKC delta by rottlerin prevented PLD2 activation in PMA-stimulated PC12 cells. Concomitant with PLD2 activation, PLD2 became phosphorylated upon PMA or bradykinin treatment of PC12 cells. Moreover, rottlerin blocked PMA- or bradykinin-induced PLD2 phosphorylation in PC12 cells. Expression of a kinase-deficient mutant of PKC delta using adenovirus-mediated gene transfer inhibited the phosphorylation and activation of PLD2 induced by PMA in PC12 cells, suggesting the phosphorylation-dependent regulation of PLD2 mediated by PKC delta kinase activity in PC12 cells. PKC delta co-immunoprecipitated with PLD2 from PC12 cell extracts, and associated with PLD2 in vitro in a PMA-dependent manner. Phospho-PLD2 immunoprecipitated from PMA-treated PC12 cells and PLD2 phosphorylated in vitro by PKC delta were resolved by two-dimensional phosphopeptide mapping and compared. At least seven phosphopeptides co-migrated, indicating the direct phosphorylation of PLD2 by PKC delta inside the cells. Immunocytochemical studies of PC12 cells revealed that after treatment with PMA, PKC delta was translocated from the cytosol to the plasma membrane where PLD2 is mainly localized. These results suggest that PKC delta-dependent direct phosphorylation plays an important role in the regulation of PLD2 activity in PC12 cells.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.     

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.     

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.     

Mechanisms of regulation of phospholipase D1 by protein kinase Calpha

It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.     

Epidermal growth factor receptor threonine and serine residues phosphorylated in vivo

The epidermal growth factor (EGF) receptor is regulated by EGF-stimulated autophosphorylation and by phorbol ester-stimulated, protein kinase C (Ca2+/phospholipid-dependent enzyme) mediated phosphorylation at identified sites. The EGF receptor contains additional phosphorylation sites including a prominent phosphothreonine and several phosphoserines which account for the majority of phosphate covalently bound to the receptor in vivo. We have identified three of these sites in EGF receptor purified from 32P-labeled A431 cells. The major phosphothreonine was identified as threonine 669 in the EGF receptor sequence. Phosphoserine residues were identified as serines 671 and 1046/1047 of the EGF receptor. Two other phosphoserine residues were localized to tryptic peptides containing multiple serine residues located carboxyl-terminal to the conserved protein kinase domain. The amino acid sequences surrounding the three identified phosphorylation sites are highly conserved in the EGF receptor and the protein products of the v-erb B and neu oncogenes. Analysis of predicted secondary structure of the EGF receptor reveals that all of the phosphorylation sites are located near beta turns. In A431 cells phosphorylation of the serine residues was dependent upon serum. In mouse B82 L cells transfected with a wild type human EGF receptor. EGF increased the 32P content in all tryptic phosphopeptides. A mutant EGF receptor lacking protein tyrosine kinase activity was phosphorylated only at threonine 669. Regulated phosphorylation of the EGF receptor at these threonine and serine residues may influence aspects of receptor function.     

Interaction of PLD1b with actin in antigen-stimulated mast cells

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Inhibition of CD3-linked phospholipase C by phorbol ester and by cAMP is associated with decreased phosphotyrosine and increased phosphoserine contents of PLC-gamma 1.

The mechanisms by which phorbol 12-myristate 13-acetate (PMA) and cAMP attenuate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) induced by ligation of the T-cell antigen receptor complex (TCR) was studied in the human Jurkat T-cell line. It has previously been shown that stimulation of Jurkat cells with antibodies to CD3, components of the TCR, elicits a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1, the predominant PLC isozyme in Jurkat cells, at multiple tyrosine residues and that such tyrosine phosphorylation leads to activation of PLC-gamma 1. Prior incubation of Jurkat cells with PMA or forskolin, which increases intracellular cAMP concentrations, prevented tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of PtdIns 4,5-P2 induced by ligation of CD3. Dose-response curves of PMA and of forskolin for the inhibition of PLC-gamma 1 tyrosine phosphorylation and of PtdIns 4,5-P2 hydrolysis were similar. These results suggest that the inhibition of PtdIns 4,5-P2 hydrolysis by PMA and cAMP is attributable to reduced tyrosine phosphorylation of PLC-gamma 1. Treatment of Jurkat cells with PMA or forskolin stimulated the phosphorylation of PLC-gamma 1 at serine 1248. PMA treatment also elicited the phosphorylation of PLC-gamma 1 at an unidentified serine site. Phosphopeptide map analysis indicated that the sites of PLC-gamma 1 phosphorylated in Jurkat cells treated with PMA and forskolin are the same as those phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), respectively. Stimulation of Jurkat cells with antibodies to CD3 also elicited phosphorylation of PLC-gamma 1 at serine 1248 and at the unidentified serine site phosphorylated in PLC-gamma 1 from PMA-treated cells. Thus, phosphorylation of PLC-gamma 1 by PKC or PKA at serine 1248 may modulate the interaction of PLC-gamma 1 with the protein tyrosine kinase or the protein tyrosine phosphatase; this altered interaction may, at least in part, be responsible for the decreased tyrosine phosphorylation of PLC-gamma 1 seen in PMA- and forskolin-treated Jurkat cells. Furthermore, in the absence of PMA, activation of PKC by diacylglycerol provides a negative feedback signal responsible for reducing the phosphotyrosine contents of PLC-gamma 1.     

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.     

Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules

The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.     

Phosphorylation of Phospholipase C‐δ1 Regulates its Enzymatic Activity

Phosphorylation of phospholipase C‐δ₁ (PLC‐δ₁) in vitro and in vivo was investigated. Of the serine/threonine kinases tested, protein kinase C (PKC) phosphorylated the serine residue(s) of bacterially expressed PLC‐δ₁ most potently. It was also demonstrated that PLC‐δ₁ directly bound PKC‐α via its pleckstrin homology (PH) domain. Using deletion mutants of PLC‐δ₁ and synthetic peptides, Ser35 in the PH domain was defined as the PKC mediated in vitro phosphorylation site of PLC‐δ₁. In vitro phosphorylation of PLC‐δ₁ by PKC stimulated [³H]PtdIns(4,5)P₂ hydrolyzing activity and [³H]Ins(1,4,5)P₃‐binding of the PLC‐δ₁. On the other hand, endogenous PLC‐δ₁ was constitutively phosphorylated and phosphoamino acid analysis revealed that major phosphorylation sites were threonine residues in quiescent cells. The phosphorylation level and the species of phosphoamino acid were not changed by various stimuli such as PMA, EGF, NGF, and forskolin. Using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry, we determined that Thr209 of PLC‐δ₁ is one of the constitutively phosphorylated sites in quiescent cells. The PLC activity was potentiated when constitutively phosphorylated PLC‐δ₁ was dephosphorylated by endogenous phosphatase(s) in vitro. Additionally, coexpression with PKC‐α reduced serine phosphorylation of PLC‐δ₁ detected by an anti‐phosphoserine antibody and PLC‐δ₁‐dependent basal production of inositol phosphates in NIH‐3T3 cells, suggesting PKC‐α activates phosphatase or inactivates another kinase involved in PLC‐δ₁ serine phosphorylation to modulate the PLC‐δ₁ activity in vivo. Taken together, these results suggest that PLC‐δ₁ has multiple phosphorylation sites and phosphorylation status of PLC‐δ₁ regulates its activity positively or negatively depends on the phosphorylation sites. J. Cell. Biochem. 108: 638–650, 2009. © 2009 Wiley‐Liss, Inc.     

Multiplicity of phosphorylation sites onEscherichia coli isocitrate dehydrogenase

Several lines of evidence indicate that the in vivo phosphorylation of isocitrate dehydrogenase (EC 1.1.1.42) inEscherichia coli occurs at multiple sites: first, the phosphorylated enzyme can be resolved by two-dimensional electrophoresis into three distinct spots differing in charge; second, the analysis of its phosphoamino acid content shows that it is modified at both serine and threonine residues; third, its extensive hydrolysis by proteolytic enzymes yields several different phosphopeptides.     

Stimulus-induced phosphorylation of PKC theta at the C-terminal hydrophobic-motif in human T lymphocytes

Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.     

Mapping of phosphorylation sites in polyomavirus large T antigen.

The phosphorylation sites of polyomavirus large T antigen from infected or transformed cells were investigated. Tryptic digestion of large T antigen from infected, 32Pi-labeled cells revealed seven major phosphopeptides. Five of these were phosphorylated only at serine residues, and two were phosphorylated at serine and threonine residues. The overall ratio of phosphoserine to phosphothreonine was 6:1. The transformed cell line B4 expressed two polyomavirus-specific phosphoproteins: large T antigen, which was only weakly phosphorylated, and a truncated form of large T antigen of 34,000 molecular weight which was heavily phosphorylated. Both showed phosphorylation patterns similar to that of large T antigen from infected cells. Peptide analyses of large T antigens encoded by the deletion mutants dl8 and dl23 or of specific fragments of wild-type large T antigen indicated that the phosphorylation sites are located in an amino-terminal region upstream of residue 194. The amino acid composition of the phosphopeptides as revealed by differential labeling with various amino acids indicated that several phosphopeptides contain overlapping sequences and that all phosphorylation sites are located in four tryptic peptides derived from a region between Met71 and Arg191. Two of the potential phosphorylation sites were identified as Ser81 and Thr187. The possible role of this modification of large T antigen is discussed.     

Extracellular signal-regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: analysis by peptide mapping.

The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) in response to insulin in Rat 1 HIRc B cells and in response to nerve growth factor (NGF) in PC12 cells has been examined. ERK1 and ERK2 are phosphorylated on serine in the absence of the stimuli and additionally on tyrosine and threonine residues after exposure to NGF and insulin. NGF stimulates tyrosine phosphorylation of ERK1 more rapidly than threonine phosphorylation. Two-dimensional phosphopeptide maps of both ERK1 and ERK2 phosphorylated in intact cells treated with NGF or with insulin display the same three predominant phosphopeptides that comigrate when digests of ERK1 and ERK2 are mixed. As many as five additional phosphopeptides are detected under certain conditions. Autophosphorylated recombinant ERK2 also contains the three tryptic phosphopeptides found in ERKs labeled in intact cells. These experiments demonstrate that ERK1 and ERK2 are phosphorylated on related sites in response to two distinct extracellular signals. The data also support the possibility that autophosphorylation may be involved in the activation of the ERKs.