Downregulation of protein kinase C‐γ is independent of a functional kinase domain

Freisewinkel, Ina; Riethmacher, Dieter; Stabel, Silvia

doi:10.1016/0014-5793(91)80307-o

Cited by 40 publications

(22 citation statements)

References 32 publications

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“…Why the results described in other recent reports (24,25) disagree with the conclusion of Ohno et al is unclear; however, the mutants used in our study provide a direct means to examine the relationship between autophosphorylation and downregulation. It should be pointed out that the studies described here measure steady-state levels of PKC protein that are influenced by the rate of de novo synthesis as well as by the rate of proteolytic degradation.…”

Section: Discussioncontrasting

confidence: 55%

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

Zhang¹,

Wang²,

Petrin³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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The autophosphorylation sites of the 12 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A. J., Paladini, R. D. & Koshland, D. E. (1990) Science 294,[408][409][410][411]. To derme the role of autophosphorylation we constructed three site-directed mutants of PKC 81i isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC 813 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a 18 isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA.Our results indicate that (1) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (it) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subceilular localization in COS cells.Protein kinase C (PKC) is a family of serine/threonine protein kinases that require diacylglycerol and phosphatidylserine for catalytic activity (1, 2). Activation of PKC plays a crucial role in the transduction ofextracellular signals leading to a variety of cellular responses-such as proliferation and differentiation, secretion of hormones and neurotransmitters, and gene expression (3)(4)(5). The importance of PKC in multistage carcinogenesis was demonstrated by the finding that the tumor-promoting phorbol esters bind to and activate PKC both in vitro and in vivo (3).Each member of the PKC family is composed of a lipidbinding regulatory domain in the NH2-terminal region and an ATP-binding catalytic domain in the COOH-terminal portion of the protein (1, 2). In vitro PKC phosphorylates many protein substrates (3); however, the relevant in vivo substrates are just beginning to be defined (6). In addition to transphosphorylation reactions, PKC autophosphorylates both in vitro and in vivo with both the regulatory and catalytic domains becoming phosphorylated (7-9).The biological role of PKC autophosphorylation has not been determined. Several studies have indicated that autophosphorylation increases th...

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Section: Discussioncontrasting

confidence: 55%

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

Zhang¹,

Wang²,

Petrin³

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…A kinase-defective form of PKC d also is susceptible to down-regulation when co-expressed with PKCg . Freisewinkel et al (1991) showed that kinase-defective PKCg also was down-regulated upon phorbol ester treatment and this, too, might be explained by activation of endogenous PKCs. Thus several PKCs may share down-regulation machinery within the cell and activation of one PKC isozyme may contribute to down-regulation of others.…”

Section: The Active Conformation Is Required For Degradation Of Pkczmentioning

confidence: 99%

“…Down-regulation of PKCa and d in membrane fractions was partially blocked by calpain inhibitor II in muscle cells (Hong et al, 1995). Kinasedefective PKCg can be down-regulated upon treatment of COS-1 cells with phorbol esters (Freisewinkel et al, 1991). Kinase-defective PKCa also can be downregulated in the presence of active PKCa in COS-1 cells (Pears and Parker, 1991).…”

Section: Introductionmentioning

confidence: 99%

Activation-dependent degradation of protein kinase Cη

et al. 2000

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Prolonged activation of protein kinase Cs (PKCs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many PKCs. To investigate the molecular mechanisms involved in the down-regulation of PKCZ, we expressed the novel PKCs Z and y and various mutant forms in baby hamster kidney cells. Upon overexpression, constitutively active PKCZ, but not wild type or kinase-dead PKCZ, underwent rapid degradation to generate several lower molecular weight polypeptides. When co-expressed with active kinases, kinase-dead PKCZ with a pseudosubstrate site mutation designed to give an active conformation was down-regulated while the wild type PKCZ was not. These results suggest requirements for kinase activity and an active conformation for down-regulation of PKCZ. Treatment with the proteasome inhibitors N-AcLeu-Leu-norleucinal and lactacystin led to accumulation of PKCZ proteolytic products and potentially ubiquitinated forms. While wild type PKCZ localizes mostly to the detergent-soluble fraction of the cell, a signi®cant portion of full-length constitutively active PKCZ and of kinase-dead, active conformation PKCZ were found in the detergent-insoluble fraction. Several proteolytic fragments of constitutively active PKCZ also were found in the detergent insoluble fraction. These full-length and proteolytic fragments of PKCZ in the detergent-insoluble fraction accumulated further in the presence of proteasome inhibitors. These data suggest that active conformation PKCZ accumulates in the detergent-insoluble compartment, is degraded by proteolysis in the presence of kinase activity, and that the cleavage products undergo further degradation via ubiquitin-mediated degradation in the proteasome.

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“…In these constructs the critical lysine at the ATP-binding site is replaced by an arginine to produce transdominant-negative phenotypes. Such PKC mutant proteins have been shown to compete with endogenous PKC in an isoenzyme specific manner (19,20,22,(31)(32)(33)(34). Employing transient transfection assays in COS-1 cells (DN) PKC-␣ K368R, (DN) PKC-⑀ K436R, and (DN) PKC-K409R have been found to lack kinase activity as recently described (19).…”

Section: Expression Of Dn Pkc-␣ K368r and Dn Pkc-⑀ K436r Mutants Blocmentioning

confidence: 99%

Conventional PKC-α, Novel PKC-ε and PKC-θ, but Not Atypical PKC-λ Are MARCKS Kinases in Intact NIH 3T3 Fibroblasts

Überall

Giselbrecht

Hellbert

et al. 1997

Journal of Biological Chemistry

108

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Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-␣ or novel nPKC-⑀ increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-␣, nPKC-⑀, and nPKC-in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-␣ K368R or (dominant negative) PKC-⑀ K436R. The fact, that the constitutively active PKC-A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-␣ and novel nPKC-⑀, but not atypical aPKC-are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.Molecular cloning and biochemical studies identified the myristoylated alanine-rich protein kinase C substrate (MARCKS) 1 as the major in vivo substrate of protein kinase C (PKC) (1-9). The ability to phosphorylate this substrate is not restricted to members of the PKC family. MARCKS is predominantly phosphorylated on serine (S) residues (in an order Ser-152 Ͼ Ser-156 Ͼ Ser-163) (10) in a PKC-dependent fashion, but can also be phosphorylated on serine and threonine residues by proline-directed protein kinases cdc2 and tau protein kinase II (11,12). MARCKS is an acidic filamentous actin cross-linking protein which is targeted to the plasma membrane by its amino-terminal, myristoylated membrane-binding domain. This specific interaction positions the substrate close to PKC, facilitating its efficient phosphorylation. One of the striking features of MARCKS is its phosphorylation-dependent translocation from the membrane to the cytosol (8). Consequently, cytosolic MARCKS is not further cross-linking actin filaments. It has also been proposed that non-phosphorylated MARCKS complexes calmodulin resulting in a reduction of Ca 2ϩ

show abstract

Downregulation of protein kinase C‐γ is independent of a functional kinase domain

Cited by 40 publications

References 32 publications

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

Characterization of site-specific mutants altered at protein kinase C beta 1 isozyme autophosphorylation sites.

Activation-dependent degradation of protein kinase Cη

Conventional PKC-α, Novel PKC-ε and PKC-θ, but Not Atypical PKC-λ Are MARCKS Kinases in Intact NIH 3T3 Fibroblasts

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