Molecular analysis of pleckstrin: the major protein kinase C substrate of platelets.
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 lambda gt11 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.
Tyers
M
M
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Haslam
R J
RJ
Rachubinski
R A
RA
Harley
C B
CB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D001798:Blood Proteins / Q000032:analysis*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D005820:Genetic Testing; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010750:Phosphoproteins; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D011493:Protein Kinase C / Q000097:blood*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured / Q000201:enzymology
D001798:Blood Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C039077:platelet protein P47; D004247:DNA; D011493:Protein Kinase C