Molecular cloning and expression of the major protein kinase C substrate of platelets

Abstract

In platelets, agonists that stimulate phosphoinositide turnover cause the rapid phosphorylation of a protein of apparent relative molecular mass (Mr) 40-47,000, called P47, by protein kinase C (PKC). Diverse identities have been ascribed to P47 including lipocortin, inositol 1,4,5-trisphosphate 5-phosphomonoesterase, pyruvate dehydrogenase alpha subunit and an actin regulatory protein. We have isolated human P47 clones by immunological screening of a lambda gt11 complementary DNA library from HL-60 cells, a human promyelocytic leukaemia cell line. P47 recombinants thus identified hybridized to a 3.0 kilobase (kb) messenger RNA in mature white blood cell lines; the same mRNA was induced in HL-60 cells during differentiation. A 1,050 base pair (bp) open reading frame that could encode a protein of Mr40,087 was confirmed by comparison with peptide sequences from platelet P47, and by expression of the putative recombinant P47 in E. coli and in vitro. The P47 sequence appears to have been conserved throughout vertebrate evolution, and is not similar to any other known sequence including human lipocortin and the alpha subunit of pyruvate dehydrogenase. The P47 protein contains a potential Ca2+-binding 'EF-hand' structure and a region that strongly resembles known PKC phosphorylation sites.

New insights into protein‐tyrosine kinase receptor signaling complexes

“…However, the number of potential interactions between signaling proteins may be increased further by another putative domain found within diverse proteins involved in intracellular signaling and/or in cytoskeletal function (Haslam et al, 1993;. Because this domain was identified initially as an internal N-and C-terminal sequence duplication in the major platelet substrate of protein kinase C, pleckstrin (Tyers et al, 1988), it has been named the pleckstrin homology, or PH, domain. In addition to pleckstrin, proteins that exhibit sequence similarity to this putative domain and that have been identified to date include the SH3 domain-binding protein 3BP-2, RasGAP and a RasGRF, the RAC family of serine/threonine protein kinases, the Drosophila and murine Sos proteins, Sec7p, the GTPase dynamin, the SH2 domain-containing protein Grb-7, oxysterol-binding protein, the yeast protein Bem3p, a trypanosomal protein kinase NrkA, the C. elegans Unc-104 kinesin-related product, the phospholipases PLCG and PLCy, and the P-spectrins (Haslam et al, 1993;.…”

Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

“…6; data not shown), both the labeled and nonlabeled cells were incubated for 5 h before treatment with various agents. Cells remained responsive to thrombin throughout the radiolabeling, as assessed by thrombin-induced shape change, aggregation, phosphorylation of myosin light chain-1 (a 20-kDa protein whose phosphorylation by a calmodulin-dependent kinase is stimulated by thrombin [11]), and phosphorylation of the 47-kDa protein kinase C substrate (identified as inositol 1,4,5-trisphosphate phosphomonoesterase [4], although doubts have been raised about this identification [24]). In some experiments (see Fig.…”

Internal amino acid sequence analysis of the 80 kDa protein kinase C substrate from rat brain: Relationship to the 87 kDa substrate from bovine brain

“…Computer-assisted searches of protein sequence databases revealed that peptides A-E did not show significant homology to any known protein. These include two previously cloned protein kinase C substrates, namely P47 from platelets [21] and the neuronal protein GAP-43 [22].…”