Mutational analysis of stress-responsive peanut dual specificity protein kinase: identification of tyrosine residues involved in regulation of protein kinase activity

Abstract

We recently reported thatArachis hypogaea serine/threonine/tyrosine (STY) protein kinase is developmentally regulated and is induced by abiotic stresses (Rudrabhatla, P., and Rajasekharan, R. (2002) Plant Physiol.130, 380-390). Other than MAPKs, the site of tyrosine phosphorylation has not been documented for any plant kinases. To study the role of tyrosines in the phosphorylation of STY protein kinase, four conserved tyrosine residues were sequentially substituted with phenylalanine and expressed as histidine fusion proteins. Mass spectrometry experiments showed that STY protein kinase autophosphorylated within the predicted kinase ATP-binding motif, activation loop, and an additional site in the C terminus. The protein kinase activity was abolished by substitution of Tyr²⁹⁷with Phe in the activation loop between subdomains VII and VIII. In addition, replacing Tyr¹⁴⁸ in the ATP-binding motif and Tyr³¹⁷ in the C-terminal domain with Phe not only obliterated the ability of the STY protein kinase protein to be phosphorylated, but also inhibited histone phosphorylation, suggesting that STY protein kinase is phosphorylated at multiple sites. Replacing Tyr²¹³ in the Thr-Glu-Tyr sequence motif with Phe resulted in a 4-fold increase in autophosphorylation and 2.8-fold increase in substrate phosphorylation activities. Mutants Y148F, Y297F, and Y317F displayed dramatically lower phosphorylation efficiency (k _cat/K _m) with ATP and histone, whereas mutant Y213F showed increased phosphorylation. Our results suggest that autophosphorylation of Tyr¹⁴⁸, Tyr²¹³, Tyr²⁹⁷, and Tyr³¹⁷ is important for the regulation of STY protein kinase activity. Our study reveals the first example of Thr-Glu-Tyr domain-mediated autoinhibition of kinases.