SUBSTRATE PHOSPHORYLATION CAPACITIES OF THE MAJOR TYROSINE PROTEIN-KINASE FROM THE HUMAN PROMYELOCYTIC CELL-LINE, HL-60

The major tyrosine protein kinase, HPK40, isolated from HL-60, the pre paration of which is described elsewhere (Ernould, A.P., Ferry, G., Ba rret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503-51 4), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid-treated enolase, actin and tubulin but not c almodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the 'src' family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corre sponding peptide RRsrc, RRLIEDNEYTARG. VALYDYESR from the SH3 domain o f pp60c-src is recognized as a substrate with a high phosphorylation l evel. DEDYIQD, derived from the phosvitin/casein kinase II, was also h ighly phosphorylated. In order to determine the minimal recognition se quence of HPK40, the phosphorylation of about 60 dito tetrapeptides wa s investigated. Some of the tetrapeptides, such as EEYE and NEYE, wer e well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS an d KYE, were recognized by HPK40, while none of the tested dipeptides w as recognized as substrate. Sequences of peptides from DRVYHPF (angiot ensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H-ras1) were exa mined as substrates. The presence of one or several acidic residues on the N-alpha-side of tyrosine residue was identified as the only appar ently favorable determinant. These results are steps towards the minim um recognition sequence, which in turn will serve as a lead for chemic al modifications in view of obtaining a specific, low-molecular-weight , inhibitor of this human tyrosine protein kinase. (C) Munksgaard 1994 .