SITE-SPECIFIC ANTI-PHOSPHOPEPTIDE ANTIBODIES - USE IN ASSESSING INSULIN-RECEPTOR SERINE THREONINE PHOSPHORYLATION STATE AND IDENTIFICATION OF SERINE-1327 AS A NOVEL SITE OF PHORBOL ESTER-INDUCED PHOSPHORYLATION/

Rabbit antisera were raised against synthetic phosphopeptides correspo nding to defined or putative sites of insulin receptor serine/threonin e phosphorylation (Ser-1305, Ser-1327, Thr-1348). All of these antibod ies bound specifically to the immuno-genic phosphopeptide but not to t he non-phosphorylated form of the peptide or to other phosphopeptides, in a microtitre plate competition enzyme-inked immunosorbent assay. A nti-PS1327 antibody reacted well with native insulin receptor prepared from phorbol ester-treated transfected CHO.T cells, but showed little reaction with receptor from untreated cells. Anti-PT1348 antibody in crude form reacted substantially with receptor from both phorbol 12-my ristate 13-acetate-treated and untreated cells, but displayed specific ity for phosphoreceptor after adsorption to remove antibodies reactive with dephosphopeptide. The ability to discriminate between receptor f rom cells treated with or without phorbol ester was retained when thes e antibodies were used to probe denatured receptor on Western blots. T hus anti-PS1327 and anti-PT1348 react with insulin receptor in a site- specific and phosphorylation-state-dependent manner. Anti-PT1348, but not anti-PS1327, also showed increased reactivity with receptor prepar ed from insulin-treated cells. The third antibody, anti-PS1305, did no t react with intact insulin receptor under any conditions. It is concl uded that serine 1327 is a major, previously unrecognized, site of pho rbol ester-induced receptor phosphorylation, and that anti-phosphopept ide antibodies will be valuable reagents with which to examine the ser ine/threonine phosphorylation state of receptor extracted from tissues .