Substrate phosphorylation capacities of the major tyrosine protein kinase from the human promyelocytic cell line, HL‐60

Abstract

The major tyrosine protein kinase, HPK40, isolated from HL‐60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214, 503–514), 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 calmodulin. 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 corresponding peptide RRsrc, RRLIEDNEYTARG. VALYDYESR from the SH3 domain of pp60c‐src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H‐rasl) were examined as substrates. The presence of one or several acidic residues on the Nα‐side of tyrosine residue was identified as the only apparently favorable determinant. These results are steps towards the minimum recognition sequence, which in turn will serve as a lead for chemical modifications in view of obtaining a specific, low‐molecular‐weight, inhibitor of this human tyrosine protein kinase.

Src kinase signaling in leukaemia

“…There are many indirect evidences that indicate an association between Src kinases and hematologic malignancies, as summarized previously (Li, 2005). (Burnett et al, 1991;Ernould, Ferry, Barret, Genton, & Boutin, 1994;Fischer et al, 1989;Lynch et al, 1993;Myers et al, 1995;Tilbrook et al, 2001;Uckun et al, 1995;Waki et al, 1994;Willman et al, 1991;Yamaguchi et al, 1997). Direct evidence for the roles of Src kinases in leukaemogenesis is inadequate; recently, we provided convincing evidence that Src kinases are required for proliferation of leukaemic cells in mice with ALL (Hu et al, 2004;Hu et al, 2006).…”

On the Organization of a Drug Discovery Platform

“…Very early on, we became interested also in kinases and some of their modifying enzymes (e.g., NMT, see above). Indeed, we started our program by choosing to explore the main tyrosine protein kinase expressed in the HL60 cancer cell line [50,51] and purification was the only option at a time when cloning was rather rare.…”