Molecular analysis of pleckstrin: The major protein kinase c substrate of platelets

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Abstract
Activation of protein kinase C (PKC) in platelets causes the immediate phosphorylation of pleckstrin, an apparent Mr 40–47,000 protein previously called 40K or P47. Pleckstrin presumably plays an important but as yet unknown role in mediating cellular responses evoked by agonist-induced phosphoinositide turnover. We have cloned the cDNA for pleckstrin from the HL-60 human promyelocytic leukemia cell line by immunological screening of a λgt11 expression library (Tyers et al.: Nature 333:470–473, 1988) and now report further analysis of the pleckstrin sequence. Pleckstrin has a deduced Mr of 40,087 and is encoded by a 1,050-bp open reading frame which is preceded by a short open reading frame that terminates before the correct initiator methionine. A single polymorphic site was found in the coding region. An unusual pattern of sequence heterogeneity occurred about a poly(A) tract in the 3′ untranslated region. The 3.0-kb pleckstrin mRNA induced upon differentiation of HL-60 cells apparently has heterogeneous 5′ ends which undergo differential regulation during HL-60 cell maturation. Analysis by multiple sequence alignment with known PKC substrates identified a strong candidate site for phosphorylation by PKC and a potential Ca2+-binding EF-hand motif. No other similarities to proteins in current databases were found.