A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

Abstract

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKCO, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC8 (67.0% in total). nPKCO has a zinc-finger-like cysteine-rich sequence (Cl region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKCO lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKCa, -13I, -1311, and -y) but not in the novel PKC subfamily (nPKC8, -e, -C, and -i). Northern (RNA) blot analyses revealed that the mRNA for nPKCO is expressed predominantly in skeletal muscle. Furthermore, nPKCO mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKCO expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKCO sequence, nPKCO was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKCO cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKCO was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKCO should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.Protein kinase C (PKC) plays a key regulatory role in a variety of cellular functions, such as cell growth and differentiation, gene expression, secretion of hormones and neurotransmitters, and membrane functions (29,30). PKC was originally identified as a serine/threonine protein kinase whose activity was dependent on calcium and phospholipids (55). Diacylglycerols (DAG), products of phosphatidylinositol turnover, lower the Ca + requirement for PKC activity from millimolar to micromolar concentrations in the presence of phosphatidylserine (PS) (18, 56), indicating that PKC is a target of DAG. Tum6r-promoting phorbol esters also bind to and activate PKC (5, 15). The pleiotropic action of phorbol esters could be explained as mimicking the activity of PKC by substituting for a physiological PKC activator, DAG. Besides the classical pathway of DAG generation through receptor-mediated hydrolysis of inositol phospholipids, recent experiments have revealed the presence of a novel pathway of DAG generation through hydrolysis of phosphatidylcholine (PC) (9). It was suggested that PKC is also involved in signaling pathways that generate DAG from PC.Recent molecular cloning experiments have established that PKC molecules consist of a protein family which can be classified into at least two major classes, conventional PKC (cPKC; isoforms a, 13I, ,II, and -y) and novel PKC (nPKC; isoforms 8, e, and t)

YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways

“…Hybridization was performed as described previously (Osada et al, 1992). The ®lters were ®nally washed with 0.16SSC (16SSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0) containing 0.1% SDS for 30 min at 658C.…”

“…RNA electrophoresis, blotting onto a nylon membrane, and hybridization were performed as described before (Osada et al, 1992). The mouse cDNA fragment (719 bp) from clone 403 was used as a hybridization probe.…”

“…COS1 cells (1610 6 cells per 10 cm dish) were transfected with the YSK1 or tagged-YSK1 expression vectors by electroporation as described before (Osada et al, 1992). After 60 h, the cells were scraped o , suspended in 200 ml of lysis bu er [20 mM Tris-HCl (pH7.5), 0.25 M sucrose, 2 mM EDTA, 0.5 mM ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), 50 mM bmercaptoethanol, 0.1% (v/v) aprotinin, 2 mM phenylmethylsulfonyl¯uoride (PMSF), 100 mg/ml leupeptin, 1 mM sodium vanadate, 0.5% Triton-X 100, 25 mM NaF] and sonicated.…”

“…The tagged-YSK1 protein was reacted with 1 ml of anti-T7 tag antibody (Novagen). Washes, treatment with the second antibody, and detection of the immunoreactive bands were carried out as reported earlier (Osada et al, 1992). The rest of the supernatant was used for autophosphorylation.…”

Identification of the primary growth response gene, ST2/T1, as a gene whose expression is differentially regulated by different protein kinase C isozymes

“…Initially, PKC was described as a Ca 2+-and phospholipid-dependent serine/threonine kinase [4,5], and later it was shown to be the major intracellular receptor for the tumorpromoting phorbol esters [6,7]. cDNA cloning and careful examination of the encoded proteins has revealed that the members of the PKC family can be classified into three major groups, namely, classical, cPKCs-c~, -/31, -1~II and -y, [8]; novel, nPKCs-~, -e, -/7, -0, and -¢t [9][10][11][12][13][14][15]; and atypical, aPKCs -5 and -t (the human counterpart of mouse aPKC-2) [10,1618]. Only the cPKCs require Ca 2+ for activity.…”