Properties and distribution of the protein inhibitor (Mr 17,000) of protein kinase C

Abstract

Ca2+-dependent hydrophobic-interaction chromatography is a powerful tool for the identification and isolation of a variety of Ca2+-binding proteins which expose a hydrophobic site(s) in the presence of Ca2+ [Gopalakrishna & Anderson (1982) Biochem. Biophys. Res. Commun. 104, 830-836; Walsh, Valentine, Ngai, Carruthers & Hollenberg (1984) Biochem. J. 224, 117-127; McDonald & Walsh (1985) Biochem. J. 232, 559-567]. Using this approach, we isolated two potent and specific protein inhibitors of protein kinase C, of 17 kDa [McDonald & Walsh (1985) Biochem. J. 232, 559-567] and 12 kDa [McDonald & Walsh (1986) Biochem. Soc. Trans. 14, 585-586]. Although these inhibitors were purified by Ca2+-dependent hydrophobic-interaction chromatography and exhibit properties similar to those of calmodulin and related Ca2+-binding proteins, we were unable to demonstrate high-affinity Ca2+ binding to these inhibitors, using equilibrium dialysis. Protein kinase C exhibited half-maximal activity at 0.6 microM-Ca2+ in the presence of phospholipid and diacylglycerol, and complete inhibition by both inhibitors was observed over the range of Ca2+ concentrations examined (10 nM-10 microM). These observations suggest that the inhibitory action of these proteins does not require Ca2+. The inclusion of proteinase inhibitors during isolation of the kinase C inhibitors, as well as two-dimensional peptide mapping and amino acid analysis of the isolated proteins, suggested that the 12 kDa inhibitor is a proteolytic fragment of the 17 kDa protein which is generated during purification. Antibodies raised in rabbits against the bovine brain 17 kDa inhibitor were shown to be specific by Western immunoblotting and the competitive enzyme-linked immunosorbent assay method and were used to study the tissue and species distribution of this protein. The inhibitor was found to be present in several bovine, murine, avian and human tissues, consistent with a role in the regulation of a variety of physiological functions involving the widely distributed protein kinase C.

The Histidine Triad Protein Hint Is Not Required for Murine Development or Cdk7 Function

“…2L). These data are in line with previous reports on the expression levels of Hint protein in the brain, stomach, and kidney (13,20). Hint expression in the adult testis continued to be most prominent in the germ cells, where the highest expression was found in the pachytene spermatocytes (unpublished data).…”

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

“…ttLeast squares fitted rms deviations (rmsd) from noncrystallographic symmetry (ncs) between protomers in the asymmetric unit. 18-23) and B (residues 71-86) and ,B elements numbered 1 (residues 31-35), 2 (residues 38-42), 3 (residues 50-56), 4 (residues 94-100), and 5 (residues 111-118). These figures were generated using SETOR (24 Mycobacterium laprae (U15187), and Saccharomyces cerevisiae (4).…”

“…This antibody also detected a protein of the expected molecular weight in primary human skin fibroblasts, mouse NIH 3T3 fibroblasts, rat 6 fibroblasts, K22 rat liver epithelial cells, bovine aortic endothelial cells, HC11 mouse mammary epithelial cells, rat intestinal epithelial cells, IEC18, and several other cell lines (data not shown). Other studies (2,5) have shown that PKCI-1 is present in a wide variety of tissues, indicating that it is expressed across a broad range of species and tissue.…”

“…A protein kinase C (PKC) inhibitory protein designated PKCI-1 (for PKC interacting protein 1) was initially purified from bovine brain as a potent inhibitor of protein kinase C (1)(2)(3)(4). Since that time, other investigators have characterized a protein similar to bovine PKCI-1 from other organisms and have not been able to demonstrate inhibitory effect on PKC activity (ref.…”

Calphobindin I (Annexin V) Inhibits Protein Kinase C.

“…Based on its characteristic actions, it is presumed that CPB I may be involved in cellular signal transduction through regulation of Ca2+ concentration, arachidonic acid cascade, and inositol phospholipid turnover. To date, several potent synthetic and naturally occurring inhibitors of PKC have been described (Hidaka and Hagiwara 1987), but there are few reports on the endogenous inhibitor of the PKC activity (McDonald et al 1987). Since CPB I is a Ca2+-and phospholipid-dependent protein like PKC, we are interested in whether CPB I affects the PKC activity.…”