These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at ..... to monitor the basal phosphothreonine content of different.
Eur J Biochem 182, 343-348 (1989) ( FEBS 1989
Antibodies directed against phosphothreonine residues as potent tools for studying protein phosphorylation Daphna HEFFETZ', Mati FRIDKIN' and Yehiel ZlCK Department of Chemical Immunology and the (Received JUIY 11, 1988/February 15, 1989)
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Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot
EJB 88 0833
Here we report the development of novel antibodies which specifically react with phosphothreonine residues [anti-(P-Thr)antibodies]. The specificity of the antibodies was assessed in radioimmunoassays where we could demonstrate that half-maximal and maximal binding of the antibodies to plates coated with BSA - P-Thr occurred at serum dilutions of 1 :4000 and 1: 1000, respectively. P-Thr inhibited antibody binding with a half-maximal effect at 40 FM. P-Ser was 200-fold less potent while P-Tyr was essentially ineffective. Anti-(P-Thr) antibodies could specifically bind to phosphothreonine-containing proteins on Western blots. Using such a procedure we could demonstrate enhanced threonine phosphorylation of the EGF receptor upon treatment of intact unlabeled A431 cells with EGF. We could further demonstrate antibodies binding to proteins present in extracts of rat hepatoma cells (Fao). P-Thr at 10 pM completely inhibited antibody binding while P-Ser, P-Tyr, Thr or Ser, each present at tenfold higher concentrations, had no such inhibitory effect. Anti-(P-Thr) antibodies were also capable of specifically immunoprecipitating 32P-labeled phosphoproteins present in Triton extracts of Fao cells. Immunoprecipitation of proteins of 38 kDa, 55 kDa, 85 kDa, 100 kDa and 155 kDa was inhibited by 1 niM P-Thr but not by P-Tyr. These findings suggest that anti-(P-Thr) antibodies could be powerful tools in studies aimed at monitoring alterations in threonine phosphorylation of specific proteins as they occur under physiological conditions in response to various extracellular stimuli. Identification of such proteins can be conveniently monitored by immunoblotting.
(c) During analysis, the 32P-labeled phosphoamino acids are partially hydrolyzed due to the need to apply either strong acidic or basic conditions. This results in low (25%) [5] and somewhat variable yields of individual phosphoamino acids, making it difficult to assess their actual content in the protein under study. (d) The relatively short half life of 32P ( z 14 days) prevents multiple analysis of samples over long time periods. (e) 32P-labeling requires preincubation of the cells in phosphate-free medium which might lead to erroneous results. Immunoblotting of cellular proteins with antibodies directed against phosphoamino acids could provide an alternative procedure for identifying specific phosphoamino acids in cellular proteins. This method is advantageous as it does not involve 32P labeling, and can therefore be employed to monitor alterations in phosphorylation of specific proteins as they occur in intact organs or even whole animals. Indeed, monoclonal [6] and polyclonal [7 - 141 antibodies directed against phosphotyrosine residues have already been generated and found useful in assessing tyrosine phosphorylation. However, tyrosine phosphorylation constitutes less than l O/O of the cellular phosphorylation reactions, while about 85% and 15% take place on serine and threonine residues, respectively Correspondence to Y. Zick, Department of Chemical Immu[5, IS]. Here we report the development of novel antibodies nology, The Weizmann Institute of Science, Rehovot IL-76100, Israel which specifically react with phosphothreonine residues [antiAbbreviarions. PtdIns, L-a-phosphatidylinositol; PtdIns4P, I
