P47 phosphoprotein of blood platelets (pleckstrin) is a major target for phorbol ester‐induced protein phosphorylation in intact platelets, granulocytes, lymphocytes, monocytes and cultured leukaemic cells: absence of P47 in non‐haematopoietic cells

Abstract

Aggregating agents including phorbol esters which activate protein kinase C induce the rapid phosphorylation of a Mr = 47,000 cytosolic protein in blood platelets (P47 or pleckstrin). This protein is well resolved by analytical 16-BAC .fwdarw. SDS two-dimensional PAGE and was purified from platelets by preparative 16-BAC .fwdarw. SDS PAGE. Polyclonal antibodies were raised to the protein in mice and rabbits. These antisera detected a single protein with the migration of P47 on Western blots of platelet extracts, and the rabbit antisera immunoprecipitated 32P-labelled P47 from platelet cytosol. The presence of P47 in other haematopoietic cells was determined by prelabelling them with 32P and observing increased 32P incorporation into the location of P47 on autoradiographs of 16-BAC .fwdarw. SDS analytical PAGE of cells exposed to phorbol ester. The identity of the phosphoprotein found in this location was further established by probing Western blots of SDS PAGE gels of cultured cells lines with the P47 antisera. P47 was detected in peripheral blood lymphocytes, monocytes and granulocytes (including the granulocytes of two unrelated patients with X-linked chronic granulomatous disease). P47 was also found in HL-60 promyelocytes (especially after differentiation with rentinoic acid). U937 histiocytes, HEL leukaemia cells, and Raji ''B'' lymphoblasts. It was not detected in normal erythrocytes, K562 leukemic cells, MOLT-3 ''T'' lymphoblasts, or in wide range of non-haematopoietic cell lines. We conclude that P47 is a major target for the action of phorbol ester induced phosphorylation in platelets, normal leucocytes and some haematopoietic cell lines. These cells have as their common feature the ability when stimulated to develop adhesive functions on their plasma membranes.