Identification of in vivo phosphorylation sites required for protein kinase D activation

Protein kinase D (PKD) is activated by phosphorylation in intact cells stimulated by phorbol esters, cell permeant diacylglycerols, bryostatin, neuropeptides, and growth factors, but the critical activating residues in PKD have not been identified. Here, we show that substitution of Ser744 and Ser748 with alanine (PKD-S744A/S748A) completely blocked PKD activation induced by phorbol-12,13-dibutyrate (PDB) treatment of intact cells as assessed by autophosphorylation and exogenous syntide-2 peptide substrate phosphorylation assays. Conversely, replacement of both serine residues with glutamic acid (PKD-S744E/S748E) markedly increased basal activity (7.5-fold increase compared with wild type PKD). PKD-S744E/S748E mutant was only slightly further stimulated by PDB treatment in vivo, suggesting that phosphorylation of these two sites induces maximal PKD activation. Two-dimensional tryptic phosphopeptide analysis obtained from PKD mutants immunoprecipitated from 32P-labeled transfected COS-7 cells showed that two major spots present in the PDB-stimulated wild type PKD or the kinase-dead PKD-D733A phosphopeptide maps completely disappeared in the kinase-deficient triple mutant PKD-D733A/S744E/S748E. Our results indicate that PKD is activated by phosphorylation of residues Ser744 and Ser748 and thus provide the first example of a non-RD kinase that is up-regulated by phosphorylation of serine/threonine residues within the activation loop.     

Mutational analysis of the potential phosphorylation sites for protein kinase C on the CCK(A) receptor

1. Many G protein-coupled receptors contain potential phosphorylation sites for protein kinase C (PKC), the exact role of which is poorly understood. In the present study, a mutant cholecystokininA (CCK(A)) receptor was generated in which the four consensus sites for PKC action were changed in an alanine. Both the wild-type (CCK(A)WT) and mutant (CCK(A)MT) receptor were stably expressed in Chinese hamster ovary (CHO) cells. 2. Binding of [3H]-cholecystokinin-(26-33)-peptide amide (CCK-8) to membranes prepared from CHO-CCK(A)WT cells and CHO-CCK(A)MT cells revealed no difference in binding affinity (Kd values of 0.72 nM and 0.86 nM CCK-8, respectively). 3. The dose-response curves for CCK-8-induced cyclic AMP accumulation and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation were shifted to the left in CHO-CCK(A)MT cells. This leftward shift was mimicked by the potent inhibitor of protein kinase activity, staurosporine. However, the effect of staurosporine was restricted to CHO-CCK(A)WT cells. This demonstrates that attenuation of CCK-8-induced activation of adenylyl cyclase and phospholipase C-beta involves a staurosporine-sensitive kinase, which acts directly at the potential sites of PKC action on the CCK(A) receptor in CCK-8-stimulated CHO-CCK(A)WT cells. 4. The potent PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), evoked a rightward shift of the dose-response curve for CCK-8-induced cyclic AMP accumulation in CHO-CCK(A)WT cells but not CHO-CCK(A)MT cells. This is in agreement with the idea that PKC acts directly at the CCK(A) receptor to attenuate adenylyl cyclase activation. 5. In contrast, TPA evoked a rightward shift of the dose-response curve for CCK-8-induced Ins(1,4,5)P3 formation in both cell lines. This demonstrates that high-level PKC activation inhibits CCK-8-induced Ins(1,4,5)P3 formation also at a post-receptor site. 6. TPA inhibition of agonist-induced Ca2+ mobilization was only partly reversed in CHO-CCK(A)MT cells. TPA also inhibited Ca2+ mobilization in response to the G protein activator, Mas-7. These findings are in agreement with the idea that partial reversal of agonist-induced Ca2+ mobilization is due to the presence of an additional site of PKC inhibition downstream of the receptor and that the mutant receptor itself is not inhibited by the action of PKC. 7. The data presented demonstrate that the predicted sites for PKC action on the CCK(A) receptor are the only sites involved in TPA-induced uncoupling of the receptor from its G proteins. In addition, the present study unveils a post-receptor site of PKC action, the physiological relevance of which may be that it provides a means for the cell to inhibit phospholipase C-beta activation by receptors that are not phosphorylated by PKC.     

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.     

Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.     

A subset of protein kinase C phosphorylation sites on the myosin II regulatory light chain inhibits phosphorylation by myosin light chain kinase

Protein kinase C (PKC) phosphorylates the regulatory light chains of smooth muscle and cytoplasmic myosin II at three known sites: S1, S2, and T9 [Ikebe, M., Hartshorne, D. J., & Elzinga, M. (1987) J. Biol. Chem. 262, 9569-9573]. Phosphorylation at these sites inhibits the actomyosin ATPase and inhibits phosphorylation of S19 on the regulatory light chain by myosin light chain kinase (MLCK) [Nishikawa, M., Sellers, J. R., Adelstein, R. S., & Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814]. To compare the effects of phosphorylation at a subset of PKC sites on the rate of MLCK phosphorylation, we substituted alanines for the known PKC phosphorylation sites in the Xenopus regulatory light chain (XRLC). PKC phosphorylation of S1A/S2A/T9A revealed secondary phosphorylation sites at T7 and T10, which are accessible both on isolated S1A/S2A/T9A and S1A/S2A/T9A-myosin hybrids. Apparent kinetic constants were determined for MLCK phosphorylation of WT XRLC and XRLC mutants: T9A, S1A/S2A, S1A/S2A/T9A, and T7A/T9A/T10A. PKC prephosphorylation of S1/2 had no effect on the rate of MLCK phosphorylation, while PKC prephosphorylation of T7/9/10 inhibited MLCK phosphorylation due to a 6-fold increase in Km. Our results suggest that phosphorylation of RLC S1/2 as observed in vivo may not be responsible for an inhibition of MLCK phosphorylation.     

Identification of specific nuclear protein kinase C isozymes and accelerated protein kinase C-dependent nuclear protein phosphorylation during myocardial ischemia

Tritium labelled (x=1.1 MBq/17.7 microg/kg) and unlabelled 8-iso-PGF2alpha (43 microg/kg) were administered intravenously to female rabbits and frequent blood and continuous urinary samples were collected up to 4 h. The total radioactivity was lost rapidly from the circulation. About 80% of the total radioactivity was found in urine within 4 h. The plasma half-life of 8-iso-PGF2alpha is found to be 1 min at the distribution phase. The terminal elimination phase half-life was about 4 min. At 1.5 min after administration 64%, 19% and 13% of the plasma radioactivity represented 8-iso-PGF2alpha, 15-keto-8-iso-PGF2alpha and beta-oxidised products, respectively. The values for 20-min plasma were 5%, 2%, and 88%. The radiochromatograms from 10 min-4 h urinary samples were dominated by more polar beta-oxidised products. Alpha-Tetranor-15-keto-13,14-dihydro-8-iso-PGF2alpha was identified as a major urinary metabolite.Thus, 8-iso-PGF2alpha metabolises in the rabbit mainly to several degraded polar metabolites through dehydrogenation at C-15, reduction of delta13-double bond and beta-oxidation, and excretes efficiently into the urine.     

Enzymatic phosphorylation of soy globulins by the protein kinase CK2. Determination of the phosphorylation sites of beta-conglycinin alpha subunit by mass spectrometry

Being quite experienced in the field of gynaecologic surgery and hysterectomies especially, being familiar with recent innovations in laparoscopic surgery and also having some own experience in laparoscopic surgery, the authors discuss the advantages and disadvantages of all surgical methods of hysterectomy. Comparing the techniques, the duration, bearing in mind the the risks, overall costs and all other surgical details, the authors concluded that laparoscopically assisted vaginal hysterectomy is the best choice because it is the least invasive, less risky, no scars are left, the postoperative recovery is quick, there are numerous indications for it, the preparation obtained as a whole can be used for further clinical examination. The only disadvantage is it is too costly and sometimes the operation itself lasts too long, so it should not be applied in some cases.     

Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, from KM,PEP >/= 2.5 mM in the basal state to 0.2 mM in the partially activated state; the KM,ATP remained virtually unchanged at approximately 1 mM, and kcat increased from 180 to 600 min-1. The high KM,PEP suggests weak binding of peptide substrates to the apoenzyme. This was confirmed by Ki > 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes in kcat and KM,ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.     

Arachidonic acid potentiates ACh receptor currents by protein kinase C activation but not by receptor phosphorylation

The effects of arachidonic acid on ACh-gated channel currents were examined using Torpedo nicotinic ACh receptors expressed in Xenopus oocytes. Arachidonic acid decreased ACh-evoked currents during treatment, to a greater extent in Ca(2+)-free extracellular solution. The currents were enhanced for more than 30 min after washing, reaching 150 and 170% in Ca(2+)-containing and -free extracellular solutions, respectively. The current enhancement was inhibited by the selective protein kinase C (PKC) inhibitor, GF109203X, whereas the current depression was not affected. Furthermore, arachidonic acid-evoked current depression was blocked in mutant ACh receptors with PKC phosphorylation site deletions on the alpha and delta subunits, but the long-lasting potentiation effect remained. These results indicate that arachidonic acid may decrease ACh receptor currents by a direct binding to PKC phosphorylation sites of the ACh receptors and may potentiate the currents via a novel pathway related to arachidonic acid-regulated PKC activation, but not via PKC phosphorylation of the ACh receptor itself.     

Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites

Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.     

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.     

Growth factors and insulin stimulate tyrosine phosphorylation of the 51C/SHIP2 protein

Antibodies raised against the 51C/SHIP2 inositol polyphosphate 5'-phosphatase were used to examine the effects of growth factors and insulin on the metabolism of this protein. Immunoblot analysis revealed that the 51C/SHIP2 protein was widely expressed in fibroblast and nonhematopoietic tumor cell lines, unlike the SHIP protein, which was found only in cell lines of hematopoietic origin. The 51C/SHIP2 antiserum precipitated a protein of approximately 145 kDa along with an activity which hydrolyzed phosphatidylinositol 3,4, 5-trisphosphate to phosphatidylinositol 3,4-bisphosphate. Tyrosine phosphorylation of the 51C/SHIP2 protein occurred in response to treatment of cells with epidermal growth (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), insulin-like growth factor-1 (IGF-1), or insulin. EGF and PDGF induced transient tyrosine phosphorylation of 51C/SHIP2, with maximal tyrosine phosphorylation occurring at 5-10 min following treatment and returning to near basal levels within 20 min. In contrast, treatment of cells with NGF, IGF-1, or insulin resulted in prolonged tyrosine phosphorylation of 51C/SHIP2 protein, with 40-80% maximal phosphorylation sustained for up to 2 h following agonist treatment. The kinetics of activation of the Akt/PKB protein kinase by the various factors correlated well with the kinetics of tyrosine phosphorylation of 51C/SHIP2. EGF, NGF, and PDGF stimulated the association of 51C/SHIP2 protein with the Shc adapter protein; however, no Shc could be detected in 51C/SHIP2-immune precipitates from cells treated with IGF-1 or insulin. The data suggest that 51C/SHIP2 may play a significant role in regulation of phosphatidylinositol 3'-kinase signaling by growth factors and insulin.     

Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase

Nyk/Mer receptor tyrosine kinase is a new member of the Ufo/Axl tyrosine kinase family and is characterized by its neural cell adhesion molecule-like extracellular domain. By using a vaccinia virus expression system to express a constitutively activated form of Nyk, we identified the major sites of Nyk autophosphorylation in tryptic peptide IY749SGDY753Y754R. Tyr-749, Tyr-753, and Tyr-754 in this peptide lie in the activation loop of the kinase domain. We also studied a series of Nyk mutants in which the three tyrosine residues were replaced individually, in pairs, or all together by phenylalanine. Single mutations of Tyr-749 or Tyr-753 to phenylalanine reduced Nyk kinase activity toward exogenous substrate to 39 or 10% of that of the wild type Nyk, respectively, whereas the Tyr-754 mutant is completely inactive. All of the double and triple Tyr-Phe mutants reduced Nyk kinase activity to a level below the background. Similar results were obtained when Nyk autophosphorylation levels were examined. Our studies suggest that full activity of Nyk/Mer kinase requires phosphorylation of all three tyrosine residues in the kinase domain (Tyr-749, Tyr-753, and Tyr-754) and that Nyk kinase activity is modulated by the level of autophosphorylation in the kinase domain. Given the highly conserved nature of this region among the Ufo/Axl receptor family members, the information presented in this report may provide insight to the biochemical properties of other members of this family.     

G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha2-adrenergic receptor subtypes

The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression. However, desensitization was imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.     

Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast two-hybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinase-inactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.