Identification of epidermal growth factor Thr-669 phosphorylation site peptide kinases as distinct MAP kinases and p34 cdc2

Abstract

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent K m ≈ 0.45 mM) for phosphorylation by purified p44 mpk , a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42 mapk . Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42 apk was measured. However, Mono Q chromatography of A431 cell extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a ∼ 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42 mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42 mapk , but it immunoreacted as a 40 kDa protein only with anti-p44 mapk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34 cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42 mapk . These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.