scholarly journals Phosphorylation of human pleckstrin on Ser-113 and Ser-117 by protein kinase C

1996 ◽  
Vol 314 (3) ◽  
pp. 937-942 ◽  
Author(s):  
Karen L. CRAIG ◽  
Calvin B. HARLEY
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Cos Cells ◽  
Phospholipid Hydrolysis ◽  
Kinase C

During platelet activation, receptor-coupled phospholipid hydrolysis stimulates protein kinase C (PKC) and results in the phosphorylation of several proteins, the most prominent being pleckstrin. Pleckstrin is composed of two repeated domains, now called pleckstrin homology (PH) domains, separated by a spacer region that contains several consensus PKC phosphorylation sites. To determine the role of PKC-dependent phosphorylation in pleckstrin function, we mapped the phosphorylation sites in vivo of wild-type and site-directed mutants of pleckstrin expressed in COS cells. Phosphorylation was found to occur almost exclusively on Ser-113 and Ser-117 within the sequence 108-KFARKS*TRRS*IRL-120. Phosphorylation of these sites was confirmed by phosphorylation of the corresponding wild-type and mutant synthetic peptides in vitro.

14-3-3 Isotypes facilitate coupling of protein kinase C-ζ to Raf-1: negative regulation by 14-3-3 phosphorylation

2000 ◽  
Vol 345 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Paulus C. J. VAN DER HOEVEN ◽  
José C. M. VAN DER WAL ◽  
Paula RUURS ◽  
Marc C. M. VAN DIJK ◽  
Wim J. VAN BLITTERSWIJK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Dependent Manner ◽  
Cos Cells ◽  
Kinase C ◽  
Pkc Ζ ◽  
Co Precipitation

14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-ζ (PKC-ζ) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-ζ-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-ζ and Raf-1 is mediated strongly by the 14-3-3β and -θ isotypes, but not by 14-3-3ζ. Far-Western blotting revealed that 14-3-3 binds PKC-ζ directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3β and Raf-1. Treatment of PKC-ζ with lambda protein phosphatase also reduced its binding to 14-3-3β in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3β facilitated PKC-ζ binding. Together, the results suggest that 14-3-3 binds both PKC-ζ (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-ζ mutant than with wild-type PKC-ζ, supporting the idea that kinase activity leads to complex dissociation. 14-3-3β and -θ were substrates for PKC-ζ, whereas 14-3-3ζ was not. Phosphorylation of 14-3-3β by PKC-ζ negatively regulated their physical association. 14-3-3β with its putative PKC-ζ phosphorylation sites mutated enhanced co-precipitation between PKC-ζ and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-ζ weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-ζ to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-ζ.

scholarly journals Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis

2005 ◽  
Vol 73 (12) ◽  
pp. 8334-8344 ◽  
Author(s):  
Ranadhir Dey ◽  
Arup Sarkar ◽  
Nivedita Majumder ◽  
Suchandra Bhattacharyya (Majumdar) ◽  
Kaushik Roychoudhury ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leishmania Donovani ◽  
Disease Process ◽  
Kinase C ◽  
Parasitic Burden ◽  
Infected Cells

ABSTRACT The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.

scholarly journals Protein kinase C-mediated desensitization of the neurokinin 1 receptor

2001 ◽  
Vol 280 (5) ◽  
pp. C1097-C1106 ◽  
Author(s):  
Olivier Déry ◽  
Kathryn A. Defea ◽  
Nigel W. Bunnett
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Neurokinin 1 Receptor ◽  
Kinase C ◽  
Naturally Occurring ◽  
Neurokinin 1 ◽  
In Cells

An understanding of the mechanisms that regulate signaling by the substance P (SP) or neurokinin 1 receptor (NK1-R) is of interest because of their role in inflammation and pain. By using activators and inhibitors of protein kinase C (PKC) and NK1-R mutations of potential PKC phosphorylation sites, we determined the role of PKC in desensitization of responses to SP. Activation of PKC abolished SP-induced Ca2+ mobilization in cells that express wild-type NK1-R. This did not occur in cells expressing a COOH-terminally truncated NK1-R (NK1-Rδ324), which may correspond to a naturally occurring variant, or a point mutant lacking eight potential PKC phosphorylation sites within the COOH tail (NK1-R Ser-338, Thr-339, Ser-352, Ser-387, Ser-388, Ser-390, Ser-392, Ser-394/Ala, NK1-RKC4). Compared with wild-type NK1-R, the t ½ of SP-induced Ca2+mobilization was seven- and twofold greater in cells expressing NK1-Rδ324 and NK1-RKC4, respectively. In cells expressing wild-type NK1-R, inhibition of PKC caused a 35% increase in the t ½ of SP-induced Ca2+mobilization. Neither inhibition of PKC nor receptor mutation affected desensitization of Ca2+ mobilization to repeated challenge with SP or SP-induced endocytosis of the NK1-R. Thus PKC regulates SP-induced Ca2+ mobilization by full-length NK1-R and does not regulate a naturally occurring truncated variant. PKC does not mediate desensitization to repeated stimulation or endocytosis of the NK1-R.

scholarly journals Specificity of protein phosphatases in the dephosphorylation of protein kinase C

1986 ◽  
Vol 240 (1) ◽  
pp. 63-67 ◽  
Author(s):  
P J Parker ◽  
J Goris ◽  
W Merlevede
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Relative Role ◽  
Phosphorylation State ◽  
Kinase C ◽  
Weak Activity ◽  
Oligomeric Forms

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.

scholarly journals Differential spatial and temporal phosphorylation of the visual receptor, rhodopsin, at two primary phosphorylation sites in mice exposed to light

2003 ◽  
Vol 374 (2) ◽  
pp. 537-543 ◽  
Author(s):  
Ryan A. ADAMS ◽  
Xinran LIU ◽  
David S. WILLIAMS ◽  
Alexandra C. NEWTON
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Outer Segment ◽  
Phorbol Esters ◽  
Phosphorylation Sites ◽  
Rod Outer Segment ◽  
Kinase C ◽  
Rhodopsin Kinase

Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser334 and Ser338, are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser338 that is evenly distributed along the rod outer segment. Phosphorylation of Ser334 is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase−/− mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser334. In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser334, with no significant effect on the phosphorylation of Ser338. Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser334, which is regulated by protein kinase C.

scholarly journals Binding of Calmodulin to the Carboxy-Terminal Region of p21 Induces Nuclear Accumulation via Inhibition of Protein Kinase C-Mediated Phosphorylation of Ser153

2005 ◽  
Vol 25 (16) ◽  
pp. 7364-7374 ◽  
Author(s):  
Aina Rodríguez-Vilarrupla ◽  
Montserrat Jaumot ◽  
Neus Abella ◽  
Núria Canela ◽  
Sonia Brun ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Localization ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Kinase C ◽  
Carboxy Terminal

ABSTRACT Intracellular localization plays an important role in the functional regulation of the cell cycle inhibitor p21. We have previously shown that calmodulin binds to p21 and that calmodulin is essential for the nuclear accumulation of p21. Here, we analyze the mechanism of this regulation. We show that calmodulin inhibits in vitro phosphorylation of p21 by protein kinase C (PKC) and that this inhibition is dependent upon calmodulin binding to p21. Two-dimensional electrophoresis analysis of cells expressing the p21 wild type or p21S153A, a nonphosphorylatable mutant of p21 at position 153, indicates that Ser153 of p21 is a phosphorylable residue in vivo. Furthermore, Western blot analysis using phospho-Ser153-specific antibodies indicates that Ser153 phosphorylation in vivo is induced when PKC is activated and calmodulin is inhibited. The mutation of Ser153 to aspartate, a pseudophosphorylated residue, inhibits the nuclear accumulation of p21. Finally, whereas wild-type p21 translocates to the cytoplasm after PKC activation in the presence of calmodulin inhibitors, p21 carrying a nonphosphorylatable residue at position 153 remains in the nucleus. We propose that calmodulin binding to p21 prevents its phosphorylation by PKC at Ser153 and consequently allows its nuclear localization. When phosphorylated at Ser153, p21 is located at the cytoplasm and disrupts stress fibers.

Sign in / Sign up

Export Citation Format

Share Document