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PubMed1992

Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein.

We mapped the in vivo phosphorylation sites for the matrix (M) protein of the Orsay and San Juan strains of vesicular stomatitis virus, Indiana serotype, using limited proteolysis and phosphoamino acid analysis. M protein was solubilized from 32P-labeled virions by using detergent and high-salt conditions, then treated with either trypsin or Staphylococcus aureus V8 protease, and analyzed by polyacrylamide gel electrophoresis and autoradiography to determine which fragments contained phosphate residues. The M protein fragment extending from amino acid 20 to the carboxy terminus contained approximately 70% of the control 32P label, while the fragment extending from amino acid 35 to the carboxy terminus had only trace amounts of label. These data indicate that the major phosphorylation site was between amino acids 20 and 34 in the Orsay strain M protein. Phosphoamino acid analysis of M protein by thin-layer electrophoresis showed the presence of phosphothreonine and phosphoserine and that phosphothreonine continued to be released after prolonged vapor-phase acid hydrolysis. These data identify Thr-31 as the primary in vivo phosphate acceptor for M protein of the Orsay strain of vesicular stomatitis virus. The San Juan strain M protein has serine at position 32, which may also be an important phosphate acceptor. In addition, phosphorylation at Ser-2, -3, or -17 occurs to a greater extent in the San Juan strain M protein than in the Orsay strain M protein. The subcellular distribution of phosphorylated M protein was investigated to determine a probable intracellular site(s) of phosphorylation. Phosphorylated M protein was associated primarily with cellular membranes, suggesting phosphorylation by a membrane-associated kinase. Virion M protein was phosphorylated to a greater extent than membrane-bound M protein, indicating that M protein phosphorylation occurs at a late stage in virus assembly. Phosphorylation of wild-type and temperature-sensitive mutant M protein was studied in vivo at the nonpermissive temperature. The data show that phosphorylated M protein was detected only in wild-type virus-infected cells and virions, suggesting that association with nucleocapsids may be required for M protein phosphorylation or that misfolding of mutant M protein at the nonpermissive temperature prevents phosphorylation.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of the hepatitis C virus NS5A protein in vitro and in vivo: properties of the NS5A-associated kinase.

NS5A derived from a hepatitis C virus (HCV) genotype 1b isolate has previously been shown to undergo phosphorylation on serine residues (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). In this report, phosphorylation of NS5A derived from HCV isolates of the 1a and distantly related 2a genotypes is demonstrated. Phosphoamino acid analysis of NS5A from the 1a isolate indicated that phosphorylation occurs predominantly on serine, with a minor fraction of threonine residues also being phosphorylated. NS5A phosphorylation was observed in diverse cell types, including COS-1, BHK-21, HeLa, and the hepatoma cell line HuH-7. Phosphorylation of a glutathione S-transferase (GST)/HCV-H NS5A fusion protein was also demonstrated in an in vitro kinase assay. This activity seemed to be highest when the pH of the reaction was neutral or slightly alkaline and displayed a preference for Mn2+ over Mg2+, with an optimum concentration of approximately 10 mM Mn2+. Somewhat surprisingly, in vitro phosphorylation of NS5A was inhibited by the addition of > or = 0.25 mM Ca2+ to reaction buffer containing Mn2+ and/or Mg2+. Comparison of phosphopeptide maps of NS5A phosphorylated in vitro and in cultured cells showed that most of the phosphopeptides comigrated, suggesting that one or more kinases involved in NS5A phosphorylation in vivo and in vitro are the same. The effects of various kinase inhibitors on NS5A phosphorylation were consistent with a kinase activity belonging to the CMGC group of serine-threonine kinases. The development of an in vitro kinase assay for NS5A phosphorylation should facilitate identification of kinase(s) responsible for its phosphorylation and of phosphorylation sites which may influence the function of NS5A in HCV propagation.

Animals↗
PubMed1996

The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases.

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.

Amino Acid Sequence↗
PubMed1999

Regulation of p53 function and stability by phosphorylation.

The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21(Waf1/Cip1)-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

Binding Sites↗
PubMed2005

Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells.

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2003

Hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites.

Hyperoxia has been shown to cause DNA damage resulting in growth arrest of cells in p53-dependent, as well as p53-independent, pathways. Although H2O2 and other peroxides have been shown to induce ataxia telangiectasia-mutated (ATM)-dependent p53 phosphorylation in response to DNA damage, the signal transduction mechanisms in response to hyperoxia are currently unknown. Here we demonstrate that hyperoxia phosphorylates the Ser15 residue of p53 independently of ATM. Hyperoxia phosphorylated p53 (Ser15) in DNA-dependent protein kinase null (DNA-PK-/-) cells, indicating that it may not depend on DNA-PK for phosphorylation of p53 (Ser15). We show that Ser37 and Ser392 residues of p53 are also phosphorylated in an ATM-independent manner in hyperoxia. In contrast, H2O2 did not phosphorylate Ser37 in either ATM+/+ or ATM-/- cells. Furthermore, H2O2 failed to phosphorylate Ser15 in ATM-/- cells. Additionally, overexpression of kinase-inactive ATM-and-Rad3-related (ATR) in HEK293T cells diminished Ser15, Ser37, and Ser392 phosphorylation compared with vector-only transfected cells. In contrast, wild-type ATR overexpression did not diminish Ser15, Ser37, or Ser392 phosphorylation. We also show that checkpoint kinase 1 (Chk1) is phosphorylated on Ser345 in response to hyperoxia, which could be inhibited by caffeine or wortmannin, potent inhibitors of phosphoinositide 3-kinase-related kinases. Hyperoxia also phosphorylated Chk1 in ATM+/+ as well as in ATM-/- cells, demonstrating an ATM-independent mechanism in Chk1 phosphorylation. Together, our data suggest that hyperoxia activates the ATR-Chk1 pathway and phosphorylates p53 at multiple sites in an ATM-independent manner, which is different from other forms of oxidative stress such as H2O2 or UV light.

Adenocarcinoma↗
PubMed1992

Phosphorylation of steroid hormone receptors.

Many observations with intact cells as well as cell-free systems suggest that receptors of the steroid hormone superfamily, along with other transcription factors, are regulated by phosphorylation. All receptors that have been analyzed carefully so far have turned out to be phosphoproteins. They are basally phosphorylated in the absence of ligand, and in many cases become hyperphosphorylated in the presence of hormone or other agonists, and sometimes of antagonists. Several studies indicate that hyperphosphorylation of receptors follows activation, and may require nuclear binding of the receptor. Serines are the predominant phosphorylated residues detected in receptors, with minor amounts of threonine. Tyrosine phosphorylation of the estrogen receptor is a subject of controversy. With various receptors, evidence has been found for phosphorylation in vivo of the N-terminal, hormone-binding, and DNA-binding domains, as well as of the hinge region. All but one of the phosphorylated sites identified in progesterone and glucocorticoid receptors by phosphopeptide mapping and sequencing are in the N-terminal domain; one is in the hinge region. Even after hormone treatment most of those sites are only partly phosphorylated, which means that several subpopulations of receptors, characterized by different states of phosphorylation and potentially different biological activities, must coexist. The majority of identified phosphorylated sites lie in consensus sequences for the PDPK. Many parallels can be discerned between phosphorylation of receptors and of other transcription factors. For example, several transcription factors become hyperphosphorylated on stimulation, and much indirect evidence points to regulation of both receptors and transcription factors by kinases and phosphatases, with cycling between different phosphorylated states. Functions of receptors that are regulated by phosphorylation are only beginning to be investigated. With transcription factors a substantial body of information is already available, and functions that appear to be thus regulated include dimerization, interactions with other proteins, binding to DNA, nuclear-cytoplasmic localization, and transcriptional activity. These and other functions may be found to be regulated by phosphorylation of receptors.

Animals↗
PubMed1991

Calcium-calmodulin and calcium-phospholipid dependent phosphorylation of membranous fraction proteins related to the tropic regulation by estradiol in the corpus luteum.

Estradiol assumes a major role in the regulation of growth, vascularization, and progesterone synthesis in the midpregnant rat corpus luteum. To explore whether molecular events triggered by estradiol could be mediated, at least in part, by protein phosphorylation, we investigated whether estradiol treatment in vivo affects endogenous luteal protein phosphorylation systems detectable in vitro. Luteal nuclear, mitochondrial, and microsomal fractions were obtained by differential centrifugation from rats hypophysectomized and hysterectomized on day 12 of pregnancy and treated with or without estradiol for 72 h. Using [gamma-32P]ATP as phosphate donor, proteins were phosphorylated in the presence or absence of either calcium (Ca), Ca plus calmodulin, or Ca plus phospholipid. Phosphoproteins were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. The Coomassie blue stained proteins and phosphoprotein profiles were markedly different in the various fractions. Estradiol treatment in vivo caused an increase in the basal endogenous phosphorylation of several proteins in vitro. It also substantially enhanced the protein kinase C (PKC) and Ca-calmodulin kinase-dependent phosphorylation of selected proteins in subcellular fractions. The Ca-calmodulin kinase catalyzed phosphorylation of microsomal 56 and 60 kilodalton (kDa) proteins was remarkably increased by estradiol. Proteins (56 and 60 kDa) were also phosphorylated when Ca-calmodulin was added to the nuclear fraction, however, this phosphorylation did not appear to be affected by estradiol treatment. A major PKC substrate in the nuclear fraction was an 80 kDa protein whose phosphorylation was increased remarkably by estradiol treatment. In the mitochondrial fraction the most striking effect of estradiol was a marked increase in PKC-mediated phosphate transfer into a 76 kDa substrate. To determine whether estradiol action on protein phosphorylation was related to its tropic effect in the corpus luteum, the hormone was administered to day 10 hypophysectomized and hysterectomized pregnant rats. In this rat model, where estradiol has no stimulatory effect on either luteal steroidogenesis or growth, neither endogenous nor kinase-mediated phosphorylation was affected by this steroid. In summary, the present investigation has revealed that in vivo treatment with estradiol affects the PKC and the Ca-calmodulin dependent in vitro phosphorylation of selected proteins localized in different subcellular compartments and further suggests that phosphorylation systems are potential control points for estradiol regulation of rat corpus luteum function.

Animals↗
PubMed1992

Prolactin-induced proliferation of the Nb2 T-lymphoma is associated with protein kinase-C-independent phosphorylation of stathmin.

Phosphorylation of stathmin, a 19-kDa protein found in many tissues, has been linked to cell differentiation and proliferation. This protein is present in lymphocytes, and both phosphorylation and expression of stathmin are regulated by lymphotropic agents. In this study an antibody specific for stathmin was used to examine phosphorylation in response to PRL. The results suggest that PRL stimulates stathmin phosphorylation in the Nb2 lymphoma and that phosphorylation correlates with PRL-induced cell proliferation. Stathmin expression does not change substantially as PRL-stimulated Nb2 cells move through the cell cycle and enter into the S-phase. Thus, stathmin phosphorylation, but not expression, is regulated by PRL. Activation of protein kinase-C (PKC) in Nb2 cells also induces phosphorylation of stathmin, but PKC does not appear to mediate phosphorylation in response to PRL. The pattern of phosphorylation in response to 12-O-tetradecanoylphorbol-13-acetate differs from that in response to PRL, and down-regulation of PKC does not inhibit PRL-induced phosphorylation or proliferation. In addition to stathmin, PRL increases phosphorylation of a group of stathmin-like proteins. Phosphorylation of these proteins also correlates well with PRL-induced proliferation. Taken together, the results suggest that phosphorylation of stathmin and stathmin-like proteins may mediate some actions of PRL in Nb2 cells. The results further suggest that activation of PKC is not an important early event in PRL-stimulated mitogenesis in Nb2 cells.

Cell Division↗
PubMed2006

The role of c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase in insulin-induced Thr69 and Thr71 phosphorylation of activating transcription factor 2.

The stimulation of cells with physiological concentrations of insulin induces a variety of responses, e.g. an increase in glucose uptake, induction of glycogen and protein synthesis, and gene expression. One of the determinants regulating insulin-mediated gene expression may be activating transcription factor 2 (ATF2). Insulin activates ATF2 by phosphorylation of Thr69 and Thr71 via a two-step mechanism, in which ATF2-Thr71 phosphorylation precedes the induction of ATF2-Thr69+71 phosphorylation by several minutes. We previously found that in c-Jun N-terminal kinase (JNK)-/- fibroblasts, cooperation of the ERK1/2 and p38 pathways is required for two-step ATF2-Thr69+71 phosphorylation in response to growth factors. Because JNK is also capable of phosphorylating ATF2, we assessed the involvement of JNK, ERK1/2 and p38 in the insulin-induced two-step ATF2 phosphorylation in JNK-expressing A14 fibroblasts and 3T3L1-adipocytes. The induction of ATF2-Thr71 phosphorylation was sensitive to MAPK kinase (MEK) 1/2-inhibition with U0126, and this phosphorylation coincided with nuclear translocation of phosphorylated ERK1/2. Use of the JNK inhibitor SP600125 or expression of dominant-negative JNK-activator SAPK kinase (SEK1) prevented the induction of ATF2-Thr69+71, but not ATF2-Thr71 phosphorylation by insulin. ATF2-dependent transcription was also sensitive to SP-treatment. Abrogation of p38 activation with SB203580 or expression of dominant-negative MKK6 had no inhibitory effect on these events. In agreement with this, the onset of ATF2-Thr69+71 phosphorylation coincided with the nuclear translocation of phosphorylated JNK. Finally, in vitro kinase assays using nuclear extracts indicated that ERK1/2 preceded JNK translocation. We conclude that sequential activation and nuclear appearance of ERK1/2 and JNK, rather than p38, underlies the two-step insulin-induced ATF2 phosphorylation in JNK-expressing cells.

3T3-L1 Cells↗
PubMed1999

Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly.

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.

Animals↗
PubMed1999

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.

Adenosine↗
PubMed1981

Filterability and pharmacology. Effects of methylxanthine derivatives on red cell phosphorylation.

Methylxanthines interact with cyclic nucleotide-dependent phosphorylation of proteins: they are potent inhibitors of cyclic nucleotide phosphodiesterase; such that they increase cAMP level and indirectly stimulate the cAMP-dependent phosphorylation. Conversely, in the course of our studies on human red blood cell protein phosphorylation we observed that methylxanthines derivatives inhibited cAMP-independent phosphorylation by casein-kinases. Intact human red blood cells were incubated in presence or inorganic 32P with and without cAMP with various concentrations of caffeine or pentoxifylline and for various times. Haemolysis was then performed and ghosts were prepared according to Dodge et al's method. Overall phosphorylation of membrane proteins was measured and SDS-PAGE was performed with the aim to determine the phosphorylation level of separate polypeptides. We observed that cAMP-dependent phosphorylations were normal but that cAMP-independent phosphorylations were decreased proportionally to the methylxanthine derivative concentration; phosphorylation of spectrin band II and protein III were decreased. Same results were observed studying phosphorylation of ghost' proteins with gamma (32P)ATP. The mechanism of this inhibition was studied using pure casein-kinase from erythrocyte cytosol, with casein or spectrin as substrates and gamma (32P)-ATP as phosphoryl donor. The results clearly showed that caffeine and pentoxifylline were competitive inhibitors of ATP in the enzymatic phosphorylation. This would be expected since the methylxanthines are adenine analogues. If the spectrin phosphorylation is necessary to the equilibrium of cytoskeleton, methylxanthine derivatives could act on erythrocyte membrane upsetting this equilibrium.

Adenosine Triphosphate↗
PubMed1999

Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

Base Sequence↗
PubMed1992

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.

Animals↗
PubMed1991

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.

Alkaloids↗
PubMed1990

A specific defect of prosolin phosphorylation in T cell leukemic lymphoblasts is associated with impaired down-regulation of DNA synthesis.

Prosolin is a major cytosolic phosphoprotein of proliferating normal PBL. Treatment of growing PBL with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)) or calcium ionophore (A23187) for 1 h caused phosphorylation of prosolin with the production of up to four prominent phosphorylated forms differing in degree of phosphorylation and/or two-dimensional electrophoretic mobility (peptides B to E). Formation of these phosphopeptides coincided with rapid down-regulation of DNA synthesis. A23187 was particularly effective in inducing phosphorylation of the more highly phosphorylated peptides D and E, suggesting the existence of a (Ca2+)-activated mechanism in their phosphorylation. The T cell leukemia cell lines Jurkat, HuT-78, CCRF-CEM, and Molt-4 showed reduced to absent ability to phosphorylate prosolin peptides rapidly in response to A23187 and also showed diminished down-regulation of DNA synthesis. In leukemic cells treated with both TPA and A23187, peptides B and C were rapidly phosphorylated, but the phosphorylation of peptides D and E seen in normal PBL remained deficient. The T cell leukemic cells appear to have intact a TPA-activated mechanism for phosphorylating prosolin peptides B and C, but share an impairment of a specific Ca2(+)-activated mechanism, possibly a Ca2(+)-dependent protein kinase, required for phosphorylation of prosolin phosphopeptides D and E. The degree of rapid down-regulation of DNA synthesis was correlated with degree of phosphorylation of peptide E in PBL and in three of four T cell leukemic cell lines. Thus, rapid phosphorylation of prosolin may mediate responses to TPA and A23187 in normal proliferating PBL, including down-regulation of DNA synthesis. A deficiency of this pathway in leukemic T cells may impede their response to physiologic growth regulatory signals utilizing this pathway and contribute to unrestrained cell growth.

Calcimycin↗
PubMed1989

Phosphorylation of the CD20 phosphoprotein in resting B lymphocytes. Regulation by protein kinase C.

CD20, a B cell integral membrane protein, regulates B cell activation and is differently phosphorylated in resting and activated cells. We have previously shown that CD20 phosphorylation is increased in activated cells and in phorbol ester-treated resting cells. Phosphorylation is also altered by agents which signal B cell proliferation, such as anti-IgM and a B cell growth factor. The present study was designed to address whether protein kinase C (PKC) or other kinases used CD20 as a substrate. When purified PKC was incubated with isolated CD20, both the 35- and 37-kDa CD20 proteins were phosphorylated in vitro. Intact resting B cells were next incubated with the protein kinase inhibitors H-7, H-8, and W-7. No change in basal CD20 phosphorylation was observed in the presence of W-7 and H-8, indicating that the protein cyclic nucleotide-dependent and calmodulin-dependent kinases were not actively phosphorylating CD20. Surprisingly, the PKC inhibitor H-7 increased CD20 phosphorylation at concentrations above 25-50 microM. To assess whether PKC either activated a phosphatase or inactivated a kinase affecting CD20 phosphorylation, tryptic phosphopeptide mapping of CD20 was performed. These studies revealed that addition of phorbol 12-myristate 13-acetate increased phosphorylation of some peptides differing from those which had increased phosphorylation following addition of H-7. Furthermore, signalling through surface immunoglobulin increased phosphorylation of CD20 peptides distinct from those hyperphosphorylated following addition of phorbol 12-myristate 13-acetate. These results demonstrate that 1) CD20 has multiple phosphorylation sites, as predicted from sequence data, and 2) whereas PKC can use CD20 as substrate, at least one other unidentified kinase phosphorylates CD20 in resting cells. Our data also predict that activation of B cells via the antigen receptor (surface IgM) may activate other protein kinases in addition to PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗