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S Jaken

Publications and source records attributed to S Jaken.

At least 37 records · Page 2Linked to original sources
PubMed1996

Protein kinase C isozymes and substrates.

As most cells express more than one type of protein kinase C (PKC), it has been difficult to establish the role of individual PKCs in cellular functions. Isozyme differences in cofactor requirements and subcellular location, in addition to variability in expression of PKCs and substrates among various cell types, are all involved in determining the effects of PKC activation. Recent identification of cellular PKC-targeting proteins and of isozyme-selective functions has provided new insight into the roles of individual PKCs in cellular processes.

Animals↗
PubMed1995

35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family.

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha-and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha-adducin. We propose naming this adducin homologue gamma-adducin.

Amino Acid Sequence↗
PubMed1995

Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. Possible molecular mechanisms.

We have previously reported that two closely related protein kinase C (PKC) isoforms, PKC alpha and PKC beta I, had divergent effects on the growth and transformation of the same parental R6 rat embryo fibroblast cell line (Housey, G. M., Johnson, M. D., Hsiao, W.-L. W. O'Brian, C. A., Murphey, J. P., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343-354; Borner, C., Filipuzzi, I., Weinstein, I. B., and Imber, R. (1991) Nature 353, 78-80). Whereas cells that overexpress PKC beta I lost anchorage dependence, grew to higher saturation densities, and generated small tumors when injected into nude mice, none of these properties were seen with cells that overexpress PKC alpha. In fact, the latter cells grew even slower and to lower saturation densities as compared to control cells. Here we investigate possible molecular mechanisms underlying the reciprocal effects of PKC alpha and PKC beta I. Overexpression of both isoforms enhanced 12-O-tetradecanoyl phorbol-13 acetate-induced expression of the growth regulatory genes c-jun, c-myc, and collagenase and enhanced feedback inhibition of epidermal growth factor receptor binding and cellular levels of diacylglycerol. However, the cells overexpressing PKC beta I differed from those overexpressing PKC alpha by displaying a decreased requirement for growth factors and by the production of a mitogenic factor. Thus, the basis for enhanced growth and transformation of cells overexpressing PKC beta I may be the establishment of an autocrine growth factor loop. These findings may be relevant to the roles of specific isoforms of PKC in carcinogenesis and tumor growth.

Animals↗
PubMed1994

Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells.

Immunocytofluorescence studies demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells but not in their SV40-transformed derivatives [Jaken et al. (1989) J. Cell Biol. 109, 697-704; Hyatt & Jaken (1990) Mol. Carcinog. 3, 45-53]. Discrete localizations imply that PKC is targeted to these areas possibly via protein-protein interactions. We have used an overlay assay to detect alpha-PKC binding proteins. The molecular interactions between alpha-PKC and the binding proteins depended on phospholipid and either calcium or phorbol esters. Unlike the kinase activity, binding activity was detected in the absence of added calcium, indicating that calcium, which is necessary for phosphorylation of most substrates, is not required for binding. Vinculin and talin, two focal contact proteins, bound alpha-PKC. REF52 cells express several annexins (I, II, and VI) which bind PKC. Both annexin I expression and vinculin expression were decreased in SV40-REF52 cells. The two major REF52 cell binding proteins (p71 and p > 200 kDa) were also down-regulated in the transformed cells, indicating transformation-sensitive regulation of PKC binding protein activity.

Amino Acid Sequence↗
PubMed1994

Protein kinase C domains involved in interactions with other proteins.

We have used a blot overlay assay to detect protein kinase C (PKC) interactions with other proteins. In many cases, the PKC binding proteins are also PKC substrates [Chapline et al. (1993) J. Biol. Chem. 268, 6858]. The purpose of the current studies was to characterize the PKC domains involved in the interactions with other proteins. alpha, beta, and epsilon isoforms of PKC interact with the same binding proteins in fibroblast cell extracts. These results indicate that constant rather than isozyme-specific (variable) regions are the major determinants of the interactions studied. PKC binding required phosphatidylserine (PS), indicating that the PS binding regulatory domain of PKC is involved in the interactions. The PKC pseudosubstrate peptide sequence, which is contained within the regulatory domain, also showed PS-dependent binding to the PKC binding proteins. To further investigate the role of the pseudosubstrate peptide in promoting PKC-protein interactions, an N-terminal truncation mutant lacking the pseudosubstrate sequence was prepared. Binding of the mutant alpha-PKC was diminished compared to wild-type alpha-PKC, although some binding was still apparent. These results indicate that the pseudosubstrate sequence contributes to, but is not the sole determinant of, PKC binding activity.

Amino Acid Sequence↗
PubMed1994

Calpain-induced downregulation of activated protein kinase C-alpha affects lung epithelial cell morphology.

A few minutes after mouse lung epithelial cell lines were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), the cells rounded up and pulled away from their neighbors. Several hours later, the cells flattened out to resume their original morphology. To begin to characterize the enzymology underlying these changes, the subcellular distribution and intracellular content of the TPA receptor, protein kinase C (PKC), and its putative endogenous regulator, the Ca(2+)-dependent protease, calpain, were investigated. Of eight PKC isozymes examined in several tumorigenic and nontumorigenic cell lines, all cells contained PKC-alpha, PKC-delta, and PKC-zeta. TPA rapidly (5 min) translocated PKC-alpha from the cytosol to the particulate fraction; PKC-alpha concentrations then decreased with continued TPA exposure. PKC-zeta levels and intracellular location were not affected. An inhibitor of PKC activity, GF 109203X, prevented the initial morphological change. The calpain II isozyme was also found in all cell lines, and its cellular content increased as a result of TPA treatment. Calpain inhibitor I did not affect the initial shape change but prevented subsequent flattening of the cells. We therefore conclude that PKC activation is required for the TPA-induced alterations in lung cell morphology and that calpain mediates the return to a flattened epithelial appearance.

Animals↗
PubMed1994

Protein kinase C isozymes in progressively transformed rat embryo fibroblasts.

The role of individual protein kinase C (PKC) isoforms in progressive transformation of a rat embryo fibroblast cell line (REF52 cells) has been evaluated. Normal (REF A) cells were transfected with SV40 to produce the partially transformed REF B cell line. REF B cells are morphologically transformed but have only limited capacity for growth in soft agar. Message and protein levels for PKC-alpha and PKC-delta were similar in REF A and B cells, indicating that expression of SV40 large T did not directly influence the amounts of PKCs. However, PKC-alpha localization was influenced. PKC-alpha was associated with focal contacts of REF A but not REF B cells, indicating that changes in location rather than content are an early event in REF52 cell transformation. Clones of REF B cells were selected for growth in soft agar (REF C cells). Levels of PKC-delta, but not PKC-alpha or epsilon, were increased in several of these clones, suggesting that increased PKC-delta content may facilitate anchorage-independent growth. In other studies, we have determined that PKCs interact with their binding proteins/substrates through their regulatory domains (RD; L. Liao et al., Biochemistry, 33: 1229-1233, 1994). These results predict that RDs could potentially inhibit PKC signaling by competing with endogenous wild-type PKCs for binding protein/substrate interactions. Overexpression of the RD of PKC-delta inhibited growth in soft agar of one representative REF C clone, whereas overexpression of the RD of PKC-alpha promoted growth in soft agar. These results suggest that RD expression may be a useful approach for dominant negative PKC inhibitors with potential isozyme specificity.

Animals↗
PubMed1994

Protein kinase C isozyme expression and down-modulation in growing, quiescent, and transformed renal proximal tubule epithelial cells.

Renal alpha-protein kinase C (PKC) is rapidly down-modulated modulated in animals treated with the renal toxin and tumor promoter, folic acid (Dong et al., Cancer Res., 53: 4542-4549, 1993). To further explore the role of PKC isozymes in renal growth and carcinogenesis, we compared phorbol ester receptor and PKC isozyme content, distribution, and regulation in primary and oncogene-altered rat renal proximal tubule epithelial cells (RPTE) in culture. Immunoblot analysis and RNase protection assays indicated that RPTE expressed at least four PKC isozymes, alpha, delta, epsilon, and zeta. Total phorbol ester receptors were decreased in primary proliferating, E1A-immortalized, and SV40-transformed RPTE compared to primary quiescent RPTE. The decrease in PDBu binding was largely due to a specific decrease in alpha-PKC protein content to approximately 50% of the level in quiescent RPTE. Degradation rates and message levels were compared to determine the mechanism for the decrease in alpha-PKC. Whereas alpha-PKC message levels in quiescent and proliferating primary RPTE were comparable, alpha-PKC degradation was increased in proliferating cells. These results indicate that the decreased alpha-PKC content was due largely to increased turnover. Phorbol ester stimulated the rate of degradation, thus demonstrating a link between degradation rate and PKC activation. These results suggest that the increased basal degradation rate in proliferating and oncogene-altered cells reflects an increase in activity of PKC in these cells.

Animals↗
PubMed1994

Protein kinase C and mammary cell differentiation: involvement of protein kinase C alpha in the induction of beta-casein expression.

Treatment of HC11 mouse mammary epithelial cells with the lactogenic hormones dexamethasone, insulin, and prolactin (DIP) leads to cellular differentiation and production of the milk protein beta-casein. The following experimental evidence suggests the involvement of protein kinase C (PKC) in DIP induced signal transduction. Down-regulation of PKC by 12-O-tetradecanoylphorbol-13-acetate or addition of CGP 41251, a selective inhibitor of PKC, inhibited beta-casein protein expression induced by DIP in HC11 cells. This inhibition occurs at the level of transcription, since the DIP mediated activation of a beta-casein promoter-luciferase reporter construct or of mammary gland specific factor (MGF), an essential transcription factor for beta-casein promoter activity, was also inhibited by CGP 41251. Inhibition or down-regulation of PKC reduced the activation of MGF by prolactin as well. PKC-alpha, the only conventional PKC isoform expressed in HC11 cells, is most likely involved in the DIP induced beta-casein expression. (a) Only PKC-alpha and PKC-epsilon are down-regulated by 12-O-tetradecanoylphorbol-13-acetate whereas PKC-delta and PKC-zeta are not. (b) Of the PKC isoforms expressed in HC11 cells, CGP 41251 inhibits PKC-alpha more potently than PKC-delta, PKC-epsilon, and PKC-zeta. The IC50 for the inhibition of beta-casein synthesis, MGF activation, and beta-casein promoter activity by CGP 41251 correlated well with the IC50 of PKC-alpha inhibition. (c) Finally, only PKC-alpha translocated to membrane fractions after DIP or prolactin treatment. Taken together, these data indicate that PKC-alpha plays an important role in the signaling pathway activated by prolactin during beta-casein induction.

Alkaloids↗
PubMed1994

Correlation between protein kinase C binding proteins and substrates in REF52 cells.

We have used a blot overlay assay to identify phosphatidylserine-dependent interactions between protein kinase C (PKC) and PKC binding proteins. The purpose of the present studies was to compare the properties of PKC binding proteins and PKC substrates detected by in vivo and in vitro phosphorylation assays. The major binding proteins and substrates in REF52 cells shared similar properties including enrichment by calmodulin-Sepharose chromatography, binding to phosphatidylserine, and resistance to heat denaturation. In addition, several of the major binding proteins and substrates were coordinately down modulated in SV40-transformed REF52 cells. The major PKC substrate, MARCKS, was also detected as a PKC binding protein. These results emphasize that the phosphatidylserine-dependent interactions between PKC and several substrates are of sufficient affinity to be detected in a blot overlay. Down modulation of binding proteins/substrates in transformed cells may reflect either decreased expression or increased basal phosphorylation of the target proteins and is likely to be involved in maintenance of the transformed phenotype.

Blotting, Western↗
PubMed1993

Regulation of protein kinase C isozymes in kidney regeneration.

Tissue damage and repair processes are important factors in renal tumor progression. To determine whether protein kinase C (PKC) is involved in these processes, we characterized PKC isozymes during rat kidney regeneration using 3 models: (a) diffuse cortical hyperplasia and hypertrophy induced by folic acid; (b) focal necrosis of the S3 segments induced by S-(1,2-dichlorovinyl)-L-cysteine; and (c) compensatory renal hypertrophy. Immunoblot analyses demonstrated that 5 PKC isozymes, alpha, beta, delta, epsilon, and zeta, were expressed in rat kidney. Six h after folic acid treatment, phorbol ester receptors were down-modulated. Down-modulation preceded an increase in DNA synthesis which was maximal at 24 h. The reduction in phorbol ester receptors was due largely to a decrease in alpha-PKC. zeta-PKC, which is not a phorbol ester receptor, was also decreased. delta- and epsilon-PKCs were not changed. However, alpha-PKC was not down-modulated during compensatory hypertrophy induced by unilateral nephrectomy. Thus, the observed decrease of alpha-PKC after folic acid treatment is most likely associated with the hyperplastic and not the hypertrophic effects of this renal toxin. These results demonstrate that activation-associated down-modulation of PKC, in particular alpha-PKC, occurs during chemical-induced renal regeneration and suggests a general role for PKC activation in non-phorbol ester tumor promotion.

Animals↗
PubMed1993

Interaction cloning of protein kinase C substrates.

We have previously used an overlay assay technique to detect proteins that interact with protein kinase C (PKC) (Hyatt, S. L., Klauck, T., and Jaken, S. (1990) Mol. Carcinogenesis 3, 45-53). In some cases, binding proteins were also identified as substrates. Therefore, we used the overlay assay approach to screen a rat kidney lambda gt11 cDNA library to isolate and identify additional PKC substrates. Two clones have now been characterized. 35A is the rat homologue of the myristoylated alanine-rich C kinase substrate (MARCKS)-related F52 cDNA, whereas 35H is a partial cDNA with substantial homology to the 3' end of beta-adducin. Both cDNAs encode proteins that bind phosphatidyl-serine (PS) and are substrates for PKC. Phosphorylation decreased both PS and PKC binding activities. Both proteins contain high density positive charge domains similar to that found in the major PKC substrate MARCKS. These results demonstrate that PKC interactions with certain substrate proteins are of sufficiently high affinity to facilitate their isolation via interaction cloning.

Amino Acid Sequence↗
PubMed1993

Transformation-sensitive localization of alpha-protein kinase C at cell-cell contacts in rat renal proximal tubule epithelial cells.

Immunocytofluorescence studies were used to compare alpha-protein kinase C (PKC) localization in primary cultures of renal proximal tubule epithelial cells (RPTE) with E1A-immortalized and SV40-transformed derivatives. Both cytosolic and nuclear staining were apparent in all of the RPTE. In primary RPTE, Triton X-100-insoluble alpha-PKC was also apparent and was concentrated in cell junctions. Cell junction alpha-PKC was diminished in E1A-immortalized and absent from SV40-transformed RPTE, even though total cellular content was not decreased. These results emphasize that subcellular location may play an important role in regulating signal transduction through PKC-dependent pathways. Phorbol ester treatment induced cell membrane ruffling in primary RPTE, and alpha-PKC was redistributed to membrane ruffles. However, the redistributed alpha-PKC was Triton soluble and, therefore, was distinct from cell junction alpha-PKC. The loss of alpha-PKC from cell junctions in phorbol ester-treated and oncogene-altered cells suggests that there are cellular determinants regulating alpha-PKC association with junctional complexes.

Animals↗
PubMed1993

Effect of alpha-protein kinase C neutralizing antibodies and the pseudosubstrate peptide on phosphorylation, migration, and growth of REF52 cells.

REF52 cells are a line of rat embryo fibroblasts that express alpha-, delta-, epsilon-, and zeta-protein kinase Cs (PKCs). In this report, we have used neutralizing antibodies to alpha-PKC to study the role of this specific PKC isozyme in REF52 cell functions. Effects of the more general PKC inhibitor, the pseudosubstrate peptide, were also studied. Previous work demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells (Jaken, S., Leach, K., and Klauck, T. J. Cell Biol., 109: 697-704, 1989). alpha-PKC redistributed to the leading lamellopodia of cells stimulated to migrate into an artificial wound, indicating that alpha-PKC activation may be coupled to migratory stimuli. The effects of the alpha-PKC neutralizing antibodies and the pseudosubstrate peptide on responses associated with focal contact functions, namely attachment, migration, and growth, were studied. The data demonstrate that the antibodies and the pseudosubstrate peptide were all efficient inhibitors of phorbol ester-stimulated phosphorylation. However, only the pseudosubstrate peptide efficiently inhibited the migration and growth responses necessary to repopulate an artificial wound. These results indicate that PKCs, but probably not alpha-PKC in particular, are important in these responses. However, because the neutralizing antibodies did not completely inhibit phorbol 12,13-dibutyrate-stimulated phosphorylation, a potential role for alpha-PKC in migration and growth cannot be excluded.

Animals↗
PubMed1992

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.

1-Phosphatidylinositol 4-Kinase↗