42] Detection and quantification of phosphotyrosine in proteins

Abstract

Publisher Summary This chapter discusses the detection and quantification of phosphotyrosine in proteins. Protein kinases can be differentiated according to their amino acid specificity. Casein kinases of type II and the cAMP-dependent kinases phosphorylate serine, while casein kinases of type I phosphorylate threonine and, to a lesser extent, serine. Another group of kinases with strict specificity for tyrosine is described in the chapter. Most tyrosine-specific protein kinase activities detected to date are implicated in cell growth control. They include the kinases associated with the cell surface receptors for two polypeptide growth factors, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), and with the transforming proteins of at least five genetically distinct groups of retroviruses. The activity of a tyrosine protein kinase in the cell is apparent from an increase in the gross level of phosphotyrosine in cell proteins. The induction of a cellular tyrosine protein kinase by growth factors or the introduction of a viral tyrosine protein kinase by infection can raise the proportion of acid-stable protein bound phosphate present as phosphotyrosine 5–10 times. Phosphatases specific for tyrosine are as yet poorly characterized, but may be inhibited by traces of Zn 2+ or by adding free phosphotyrosine or analogs. The simplest strategy is to lyse cells directly into a denaturing solution, or to stop an in vitro reaction with denaturing agents.