Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Baxter, Gregory T.; Oto, Edwin; Daniel-Issakani, Sarkiz; Strulovici, Berta

doi:10.1016/s0021-9258(18)46033-4

Cited by 51 publications

(14 citation statements)

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“…Second, they co-migrate in SDS-polyacrylamide gels when mixed . Third, both cell types contain a 90-and a 40-kD PKCe immunoreactive species, which are related if not identical as was shown by peptide mapping (Baxter et al ., 1992) . Here we add a new criterion, namely, physical association with CK8/18.…”

Section: Discussionsupporting

confidence: 54%

“…For example, a catalytic 40-kD PKCe immunoreactive fragment has been noted in the human small cell lung carcinoma (SCLC) cell line NCI-N417. The level of this 40-kD species increased after treating cells with phorbol myristate acetate (TPA) or gastrin-releasing peptide (Baxter et al, 1992) . The 40-kD species noted in the NCI-N417 cells may represent a transcript variant (Schaap et al, 1989(Schaap et al, , 1990, an alternate gene product (Ono et al ., 1988) or a proteolytically activated fragment of the parent PKCe.…”

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confidence: 99%

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PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

Omary

Baxter

Chou

et al. 1992

The Journal of Cell Biology

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Abstract. A 40-kD protein kinase C (PKC)E related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18. The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKCE specific antibody showed that the 40-kD species, and not native PKCE (90 kD) associated with the cytokeratins. Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKCE, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. C YTOKERATINS (CK)' are a group of intermediate filament (IF) proteins which are expressed primarily in epithelial tissues (Lazarides, 1982 ;Steinert and Roop, 1988 ;Franke et al ., 1981;Osborn and Weber, 1986) . The 30 or so polypeptides which make up the family ofcytokeratin proteins are divided into acidic (type I) and basic/ neutral (type II) keratins. In epithelial cells, CK are found as mosaic noncovalent polymers with an assembly consisting of at least one type I and one type II CK (Steinert and Roop, 1988). For example, "simple" single layer epithelial cells such as intestinal epithelia express CK8 (type II) and CK18 (type I), whereas esophageal epithelial cells express CK4 (type II) and CK13 (type I) predominantly. Cytokeratins are not only important as tissue-specific markers, they also form important markers of cell differentiation .IF proteins, including cytokeratins, undergo several posttranslational modifications such as N112-terminal acetylation (Steinert and Idler, 1975), glycosylation (King and Hounsell, 1989;Roberts and Brunt, 1986), and serine/threonine phosphorylation (Steinert, 1988;Gilmartin et al ., 1984 ;Yeagle et al., 1990;Baribault et al., 1989) . The functional role of IF protein phosphorylation is not well under- A peptide pseudosubstrate specific for PKCE inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates . Tryptic peptide map analysis -of the cytokeratins that were phosphorylated by purified rat brain PKCE or as immunoprecipitates by the associated kinase showed similar phosphopeptides . Furthermore, PKCE immunoreactive species and CK8/18 colocalized using immunofluorescent double staining . We propose that a kinase related to the catalytic fragment of PKCE physically associates with and phosphorylates cytokeratins 8 and 18.

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Section: Discussionsupporting

confidence: 54%

mentioning

confidence: 99%

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

Omary

Baxter

Chou

et al. 1992

The Journal of Cell Biology

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“…To further characterize the kinase, lysates of WISH cells incubated for 24 h without or with 10 -7 M PMA were subjected to immunocapture and phosphorylation. A member of the PKC family, PKC e, known to be associated with CK8 and CK18 (Omary et al, 1992), was shown to be resistant to down-regulation by PMA treatment (Baxter et al, 1992). As seen in Fig.…”

Section: Ldenafication Of the Components Of The Upar Complexmentioning

confidence: 83%

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.

Busso

Masur

Lazega

et al. 1994

The Journal of Cell Biology

252

175

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A human epithelial cell line, WISH, and a mouse cell line, LB6-uPAR, transfected with the human urokinase receptor (uPAR), both expressed high affinity uPAR but undetectable levels of urokinase (uPA). In two independent assays, binding of exogenous pro-uPA produced an up to threefold enhancement of migration. The migration was time and concentration dependent and did not involve extracellular proteolysis. This biologic response suggested that uPAR can trigger an intracellular signal. Since this receptor is a glycosyl-phosphatidylinositol-linked protein, we postulated that it must do so by interacting with other proteins, among which, by analogy to other systems, would be a kinase. To test this hypothesis, we carried out a solid phase capture of uPAR from WISH cell lysates using either antibodies against uPAR or pro-uPA adsorbed to plastic wells, followed by in vitro phosphorylation of the immobilized proteins. SDS-PAGE and autoradiography revealed two phosphorylated protein bands of 47 and 55 kD. Both proteins were phosphorylated on serine residues. Partial sequence of the two proteins showed a 100% homology to cytokeratin 18 (CK18) and 8 (CK8), respectively. A similar pattern of phosphorylation was obtained with lysates from A459 cells, a lung carcinoma, but not HL60, LB6-uPAR or HEp3 cell lysates, suggesting that the identified multiprotein uPAR- complex may be specific for simple epithelia. Moreover, immunocapture with antibody to another glycosyl-phosphatidylinositol-linked protein, CD55, which is highly expressed in WISH cells, was ineffective. The kinase was tentatively identified as protein kinase C, because it was inhibited by an analogue of staurosporine more specific for PKC and not by a PKA or tyrosine kinase inhibitors. The kinase was tentatively identified as PKC epsilon because of its resistance to PMA down- modulation, independence of Ca2+ for activity, and reaction with a specific anti-PKC epsilon antibody in Western blots. Cell fractionation into cytosolic and particulate fractions revealed that all four proteins, the kinase, uPAR, CK18, and CK8, were present in the particulate fraction. In vivo, CK8, and to a lesser degree CK18, were found to be phosphorylated on serine residues. Occupation of uPAR elicited a time-dependent increase in the phosphorylation intensity of CK8, a cell shape change and a redistribution of the cytokeratin filaments. These results strongly suggest that uPAR serves not only as an anchor for uPA but participates in a signal transduction pathway resulting in a pronounced biological response.

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“…PKC protein fragment-derived peptide inhibitors: As mentioned above, the regulatory region of PKCs contains an autoinhibitory pseudosubstrate domain that interacts with the C4 domain. These pseudosubstrate-derived peptides are used as selective and cell-permeable inhibitor of PKC, such as PKCα/β pseudosubstrate peptide (PKC19-36) (RFARKGALRQKNVHEVKN) and its derivative (FARKGALRQ) [128,133], PKCε pseudosubstrate peptide (ERMRPRKRQGAVRRRV) [134], and PKCζ pseudosubstrate-derived ζ-inhibitory peptide (ZIP; SIYRRGARRWRKL) [135]. Although these PKC protein fragmentderived peptide inhibitors are a useful tool for understanding the PKC-mediated signaling pathway, they may not be suitable as therapeutic agents because of their weak inhibitory abilities for PKC [2].…”

Section: Peptide Inhibitorsmentioning

confidence: 99%

Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials

et al. 2021

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Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinase, is classed into three subfamilies based on their structural and activation characteristics: conventional or classic PKC isozymes (cPKCs; α, βI, βII, and γ), novel or non-classic PKC isozymes (nPKCs; δ, ε, η, and θ), and atypical PKC isozymes (aPKCs; ζ, ι, and λ). PKC inhibitors and activators are used to understand PKC-mediated intracellular signaling pathways and for the diagnosis and treatment of various PKC-associated diseases, such as cancers, neurological diseases, cardiovascular diseases, and infections. Many clinical trials of PKC inhibitors in cancers showed no significant clinical benefits, meaning that there is a limitation to design a cancer therapeutic strategy targeting PKC alone. This review will focus on the activators and inhibitors of PKC and their applications in clinical trials.

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Constitutive presence of a catalytic fragment of protein kinase C epsilon in a small cell lung carcinoma cell line.

Cited by 51 publications

References 50 publications

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

PKC epsilon-related kinase associates with and phosphorylates cytokeratin 8 and 18.

Induction of cell migration by pro-urokinase binding to its receptor: possible mechanism for signal transduction in human epithelial cells.

Activators and Inhibitors of Protein Kinase C (PKC): Their Applications in Clinical Trials

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