Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.     

Identification of a novel annexin in Hydra vulgaris. Characterization, cDNA cloning, and protein kinase C phosphorylation of annexin XII.

As a first step toward the elucidation of a simple animal model in which to investigate annexin function, we identified, isolated, and characterized a novel annexin from Hydra vulgaris, annexin XII. A hydra cDNA library was screened using a probe generated by polymerase chain reaction from primers based on the partial amino acid sequence of annexin XII. Annexin XII cDNA was cloned and the functional protein was expressed in high yields in Escherichia coli. The annexin XII cDNA sequence predicted a 316-amino acid protein that had between 44 and 54% sequence identity with the Ca2+-binding core domains of previously characterized vertebrate and Drosophila annexins. The amino-terminal domain of annexin XII did not have sequence similarity with other known annexins except at and around a site that resembled known protein kinase C (PKC) phosphorylation sites in other annexins. As anticipated from its sequence, annexin XII was a high affinity substrate for purified rat brain PKC; half-maximal phosphorylation occurred below 0.1 microM annexin XII, and incorporation of up to 0.8 mol of phosphate/mol of annexin XII was observed. A PKC-like activity in hydra extracts also phosphorylated annexin XII. In summary, hydra promises to be a valuable model system for investigating the biological function of annexins and for determining how this function is modulated by PKC phosphorylation.     

Activation of annexin 7 by protein kinase C in vitro and in vivo

Annexin 7, a Ca(2+)/GTP-activated membrane fusion protein, is preferentially phosphorylated in intact chromaffin cells, and the levels of annexin 7 phosphorylation increase quantitatively in proportion to the extent of catecholamine secretion. Consistently, various protein kinase C inhibitors proportionately reduce both secretion and phosphorylation of annexin 7 in these cells. In vitro, annexin 7 is quantitatively phosphorylated by protein kinase C to a mole ratio of 2.0, and phosphorylation is extraordinarily sensitive to variables such as pH, calcium, phospholipid, phorbol ester, and annexin 7 concentration. Phosphorylation of annexin 7 by protein kinase C significantly potentiates the ability of the protein to fuse phospholipid vesicles and lowers the half-maximal concentration of calcium needed for this fusion process. Furthermore, other protein kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein-tyrosine kinase pp60(c-)(src), also label annexin 7 with high efficiency but do not have this effect on membrane fusion. In the case of pp60(c-)(src), we note that this kinase, if anything, modestly suppresses the membrane fusion activity of annexin 7. These results thus lead us to hypothesize that annexin 7 may be a positive mediator for protein kinase C action in the exocytotic membrane fusion reaction in chromaffin cells.     

Calphobindins (placental annexins) inhibit protein kinase C.

Calphobindins (CPBs, placental annexins) are intracellular Ca(2+)- and phospholipid-dependent proteins like protein kinase C [EC 2.7.1.37]. We investigated the inhibitory effects of calphobindins on the protein kinase C activity in vitro. CPB I inhibited the protein kinase C activity for both histone phosphorylation and lipocortin phosphorylation, but CPB II and CPB III inhibited only the protein kinase C activity for histone phosphorylation. In the case of histone phosphorylation, all CPBs inhibited the protein kinase C activity in a concentration-dependent manner, and the IC50 (concentration required for 50% inhibition) value of CPB I was 70 nM. The inhibition of protein kinase C by CPB I was Ca(2+)-dependent, and did not disappear upon increasing the concentration of phosphatidyl-serine. Kinetic analysis by double-reciprocal plots indicated that CPB I interacted not only with phosphatidylserine but also with protein kinase C. Although CPB I partially interacts with phospholipid, it is conceivable that the inhibitory action of CPB I on protein kinase C results from direct interaction of CPB I with protein kinase C. Since CPBs are mainly present under the plasma membrane, it is presumed that CPB I is an endogenous inhibitor of protein kinase C, and according to intracellular circumstances, CPB II and CPB III may also be endogenous inhibitors.     

Regulation of the chromaffin granule aggregating activity of annexin I by phosphorylation.

Annexin I (lipocortin I) binds to secretory granule membranes and promotes their aggregation in a Ca(2+)-dependent manner [Creutz, C. E., et al. (1987) J. Biol. Chem. 262, 1860-1868; Drust, D. S., & Creutz, C. E. (1988) Nature 331, 88-91]. It is also phosphorylated on serine residues when bovine chromaffin cells are stimulated to secrete [Michener, M. L., et al. (1986) J. Biol. Chem. 261, 6548-6555], suggesting phosphorylation may be involved in modulating the function of annexin I. We report here that phosphorylation of the N-terminal tail by protein kinase C strongly inhibits the ability of annexin I to aggregate chromaffin granules by increasing the calcium requirement 4-fold. This inhibition was readily reversed when the protein was dephosphorylated by protein phosphatase 2A. The inhibition was not due to inability of phosphorylated annexin I to bind to chromaffin granules, since the phosphorylated form bound to the granule membrane at slightly lower levels of calcium than the native form. The phosphorylated annexin I also bound to 20% phosphatidylserine/80% phosphatidylcholine vesicles at lower Ca2+ levels than the native form. The inhibitory effect of phosphorylation on the granule aggregating activity of annexin I was found to be amplified by an unusual mechanism: The phosphorylated form inhibited the activity of the unphosphorylated form. The possible importance of the regulation of annexin I activity by phosphorylation in exocytosis is discussed.     

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.     

Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine

We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.     

Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca(2+) concentrations and oscillatory association of PKCβ-enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca(2+) mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca(2+) entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.     

Protamine induces autophosphorylation of protein kinase C: stimulation of protein kinase C-mediated protamine phosphorylation by histone.

Protein kinase C (PKC), a protein phosphorylating enzyme, is characterized by its need for an acidic phospholipid and for activators such as Ca2+ and diacylglycerol. The substrate commonly used in experiments with PKC is a basic protein, histone III-S, which needs the activators mentioned. However, protamine, a natural basic substrate for PKC, does not require the presence of cofactor/activator. We report here that protamine can induce the autophosphorylation of PKC in the absence of any PKC-cofactor or activator; this may represent a possible mechanism of cofactor-independent phosphorylation of this protein. It was investigated if protamine itself can act as a PKC-activator and stimulate histone phosphorylation in the manner of Ca2+ and phospholipids. Experiments however showed that protamine is not a general effector of PKC. On the contrary, histone stimulated PKC-mediated protamine phosphorylation and protamine-induced PKC-autophosphorylation. Histone alone did not induce PKC-autophosphorylation. Kinetic studies suggest that histone increases the maximal velocity (Vmax) of protamine kinase activity of PKC without affecting the affinity (Km). Other polycationic proteins such as polyarginine serine and polyarginine tyrosine were not found to influence PKC-mediated protamine phosphorylation, indicating that the observed effects are specific to histone, and are not general for all polycationic proteins. These results suggest that histone can modulate the protamine kinase activity of PKC by stimulating protamine-induced PKC-autophosphorylation.     

Muscarinic activation of mitogen-activated protein kinase in rat thyroid epithelial cells

Carbachol (Cch), a muscarinic acetylcholine receptors (mAChR) agonist, produces time- and dose-dependent increases in mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) phosphorylation in nondifferentiated Fischer rat thyroid (FRT) epithelial cells. Cells pretreatment with the selective phospholipase C inhibitor U73122 resulted in a decrease of Cch-stimulated ERK1/2 phosphorylation. These data indicated that the effect of mAChR on ERK activation could be mediated through agonist-induced Ca(2+) mobilization or PKC activation. Phosphorylation of ERK1/2 was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate acetate (PMA), but was not altered either by PKC inhibitor GF109203X or by down-regulation of PKC. Phosphorylation of ERK1/2 was elevated by a direct [Ca(2+)](i) increase caused by thapsigargin or ionophore. Additionally, Cch-induced ERK1/2 phosphorylation was reduced after either inhibition of Ca(2+) influx or intracellular Ca(2+) release. Nevertheless, Cch-mediated ERK1/2 activation was genistein sensitive, indicating the involvement of protein tyrosine kinases on the downstream signalling of mAChR. Pretreatment of the cells with PP2 markedly decreased Cch-induced ERK1/2 phosphorylation, suggesting a role of Src family of tyrosine kinases in the signal transduction pathway involved in ERK1/2 activation by mAChR. To test the biological consequences of ERK activation, we examined the effect of mAChR on cell functions. Cch stimulation of FRT cells did not affect cell proliferation, but increased protein synthesis. This effect was significantly attenuated by PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK). This study demonstrated that muscarinic receptor-mediated increase in the ERK1/2 phosphorylation was dependent on [Ca(2+)](i) but independent of PKC and was mediated by the Src family of tyrosine kinases. Our results also supported the idea that the protein synthesis stimulated by mAChR in polarized FRT epithelial cells was regulated by the ERK1/2 phosphorylation pathway.     

Site-specific mutagenesis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding.

Annexin V (placental anticoagulant protein I) binds tightly to anionic phospholipid vesicles in the presence of calcium. Four mutant proteins were expressed in Escherichia coli in which Ala replaced one of the following residues in the third repeat of annexin V: Arg-200, His-204, Arg-206, or Lys-207. In a competitive fluorescence quenching assay, the wild-type recombinant protein had the same affinity for phosphatidylserine-containing vesicles as the placentally derived protein. The affinity of the four mutant proteins for phosphatidylserine-containing vesicles was unchanged relative to wild-type protein. We conclude that His-204 and adjacent basic residues, including the highly conserved Arg-200 residue, are not required for high-affinity phospholipid binding.     

Phosphorylation of annexin A1 by TRPM7 kinase: a switch regulating the induction of an α-helix

TRPM7 is an unusual bifunctional protein consisting of an α-kinase domain fused to a TRP ion channel. Previously, we have identified annexin A1 as a substrate for TRPM7 kinase and found that TRPM7 phosphorylates annexin A1 at Ser5 within the N-terminal α-helix. Annexin A1 is a Ca(2+)-dependent membrane binding protein, which has been implicated in membrane trafficking and reorganization. The N-terminal tail of annexin A1 can interact with either membranes or S100A11 protein, and it adopts the conformation of an amphipathic α-helix upon these interactions. Moreover, the existing evidence indicates that the formation of an α-helix is essential for these interactions. Here we show that phosphorylation at Ser5 prevents the N-terminal peptide of annexin A1 from adopting an α-helical conformation in the presence of membrane-mimetic micelles as well as phospholipid vesicles. We also show that phosphorylation at Ser5 dramatically weakens the binding of the peptide to S100A11. Our data suggest that phosphorylation at Ser5 regulates the interaction of annexin A1 with membranes as well as S100A11 protein.     

Inhibition of calcium signaling in ultraviolet-irradiated fibroblasts: role of tyrosine phosphorylation and protein kinase C.

Our aim was to study whether ultraviolet radiation produced any alterations in the subsequent signaling response of V79 fibroblasts to mitogenic stimulus. In ultraviolet C (UVC)-irradiated V79 fibroblasts, increase in cytosolic calcium in response to thrombin was nearly abolished in the presence of 3 mM external Ca(2+). UVC-treated V79 cells showed a greatly enhanced permeability to Ca(2+) which was reversed by pretreatment with genistein, a tyrosine kinase inhibitor. Genistein also alleviated the inhibition of thrombin response caused by UVC. In UVC-treated cells, significant activation of protein kinase C (PKC) occurred only on exposure to 3 mM external calcium and PKC inhibitors (H-7 or staurosporine) reversed UVC-induced adverse effects on the thrombin response. Therefore, it is likely that protein tyrosine phosphorylation by UVC may play a role in the subsequent inhibition of thrombin response in V79 cells through increased calcium influx and activation of PKC.     

Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes.

Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) was compared with calcium/phosphatidylserine (Ca/PS). The substrate specificity of PKC was more limited with PS/PMA. Substrates could be divided into three overlapping groups according to their relative level of phosphorylation: C1, relatively preferred substrates with Ca/PS, included dephosphin, histone, and peptide GS1-10. C2, relatively preferred with PS/PMA, included myelin basic protein and MARCKS. C3, substrates independent of activators. PS/PMA altered the Vmax of PKC for substrate, and decreased the Km for Mg2+. Differential substrate phosphorylation by PS/PMA also occurred for PKC isozymes resolved by hydroxylapatite chromatography and was most dramatic for PKC-alpha, which could no longer phosphorylate histone or GS1-12. Differential activities of PKC were also observed in synaptosol and in intact synaptosomes where PMA stimulated phosphorylation of MARCKS, but not dephosphin. It was further shown that dephosphin was indeed a substrate of PKC in the intact synaptosomes by use of a repolarization-dependent dephosphin phosphorylation assay. The differential PKC activities could also be distinguished by inhibitors. H-7 was equipotent, palmitoylcarnitine did not inhibit in vitro C2 phosphorylation, but inhibited dephosphin in intact synaptosomes, and sphingosine did not inhibit C1 substrates and was without effect on dephosphin in intact synaptosomes. Therefore PS/PMA alters or limits the substrate specificity of PKC, leading to a differential substrate phosphorylation in vitro and in intact synaptosomes and differential inhibitor sensitivity. The pattern of protein phosphorylation observed after PKC activation in intact cells will therefore be dependent upon the activator.     

A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.     

Inhibition of protein kinase C by a peptide conjugate homologous to a domain of the retroviral protein p15E

Retroviral infection is associated with immunosuppression, which has been shown to be due, in part, to the action of the envelope protein p15E. We studied a synthetic peptide (CKS-17) homologous to a highly conserved domain of the retroviral envelope protein p15E, which, when conjugated to BSA (CKS-17-BSA), can inhibit IL-1- and phorbol ester-mediated responses in cultured murine thymoma cells, and Ca2(+)- and phosphatidylserine-dependent protein kinase C (PKC) activity of cell homogenates. We characterized the mechanism of inhibition of PKC by the peptide. Using PKC purified from rat brain we found that CKS-17-BSA inhibited PKC-catalyzed Ca2(+)- and phosphatidylserine-dependent histone phosphorylation with an estimated ID50 of 4 microM. CKS-17-BSA did not inhibit the catalytic subunit of cAMP-dependent protein kinase. CKS-17-BSA also inhibited the Ca2(+)- and PS-independent activity of a catalytic fragment of PKC that was generated by limited trypsin treatment. However, CKS-17-BSA did not act as a competitive inhibitor of PKC with respect to ATP or phosphoacceptor substrate, despite the similarity between the CKS-17 sequence and substrates and pseudosubstrates of PKC. We conclude that this peptide homologue of a retroviral envelope protein has a novel mechanism of inhibition of PKC.     

Phosphorylation of coagulation factor II by phospholipid/Ca(2+)-dependent protein kinase (protein kinase C)

Prothrombin is a major constituent of the blood coagulation cascade and requires phospholipid and Ca2+ for its activation. We have found that phospholipid/Ca(2+)-dependent protein kinase (Protein kinase C) phosphorylates prothrombin and the associated apparent Km value for prothrombin (0.86 microM) is comparable to the Km value reported for most known substrates of protein kinase C. A 2-dimension separation analysis revealed that serine residue was apparently phosphorylated by PKC. The phosphorylation was inhibited by such phosphatidylserine- and/or Ca2+ competitive protein kinase C inhibitors as trifluoperazine, palmitoylcarnitine and gossypol. These results suggest that protein kinase C phosphorylation was involved in the regulation of blood coagulation.     

Nonsteroidal antiestrogen suppresses protein kinase C--its inhibitory effect on interaction of substrate protein with membrane.

The mechanism by which nonsteroidal antiestrogen inhibits Ca(2+)- and phospholipid-dependent protein kinase (PKC) activity was investigated. Antiestrogenic agents, clomiphene and tamoxifen, inhibited the PKC-dependent phosphorylation of histone and r-annexin I in a dose-dependent manner. Ki values for the agents were different for two substrate proteins. The inhibitory action of the agents depended on the membrane-substrate protein interaction. Phosphorylation of cytoplasmic proteins obtained from rat uterus and mammary gland, including annexin I, by endogenous PKC was also inhibited by low concentrations of these agents. These results suggest that the inhibitory action of nonsteroidal antiestrogens occurs through their inhibitory effect on the membrane-substrate protein interaction.