Importance of threonine residues in the regulation of peanut serine/threonine/tyrosine protein kinase activity

Abstract

Protein tyrosine phosphorylation is carried out by dual-specificity kinases in plants. Peanut dual-specificity kinase has been shown to be regulated by tyrosine phosphorylation. However, the role of threonine residues in the regulation of peanut serine/threonine/tyrosine (STY) protein kinase is not yet documented. In the present study, we have investigated the role of threonine residues in the regulation of peanut STY protein kinase activity. The four threonine residues in the kinase activation loop and Thr²¹¹ in the threonine-glutamate-tyrosine (TEY) motif were mutated to alanine to study their role in the regulation of kinase activity. The protein kinase activity was abolished when Thr²¹¹ of TEY motif and Thr²⁹⁶ of activation loop were converted to alanine, suggesting that they positively regulate the kinase activity. The ability of T211A and T296A to phosphorylate histone H1 was also reduced drastically. The other mutants T287A, T291A and T294A did not show any change in their ability to autophosphorylate or phosphorylate histone H1 when compared to wild-type peanut STY protein kinase. Data presented here suggests the importance of threonine residues in the regulation of peanut STY protein kinase activity and emphasizes the complexity of regulation of dual-specificity protein kinases in plants.

Introduction

Protein kinases catalyze the transfer of γ-phosphate from ATP to serine, threonine or tyrosine residues in target proteins. Protein tyrosine phosphorylation plays an important role in growth, development and regulation of metabolic processes in animals. The role of receptor and non-receptor tyrosine kinases has been studied extensively in animals systems. Presence of a classical protein tyrosine kinase in plant species is still controversial. However, the phosphorylation of tyrosine residues has been shown to occur in various plants. Tyrosine phosphorylation was shown to occur during coconut zygotic embryo development [1]. It was also proposed to play a role in plant development and embryogenesis [2]. Phosphorylation on tyrosine residues also plays an important role in plant movements and the degree of petiole bending is well correlated with tyrosine phosphorylation on actin molecules in Mimosa pudica [3]. Tyrosine phosphorylation was also documented in Catharanthus roseus roots that were transformed by Agrobacterium rhizogenes [4]. Recently, the involvement of protein tyrosine phosphorylation was documented during phytohormone-stimulated cell proliferation in Arabidopsis hypocotyls [5]. Although tyrosine phosphorylation is well documented in plant systems, bona fide tyrosine kinase has not been identified so far.

Earlier attempts to clone a plant tyrosine kinase resulted in the identification of only dual-specificity protein kinases that phosphorylate on serine, threonine and tyrosine residues [6], [7]. We have earlier shown that the peanut dual-specificity protein kinase is stress responsive and developmentally regulated during seed development [7]. Genome-wide analysis of Arabidopsis using the delineated tyrosine kinase motif from animals revealed the presence of only dual-specificity kinases, raising an intriguing possibility that plants lack classical tyrosine kinases [8]. This class of dual-specificity kinases was known to undergo intramolecular type of phosphorylation mechanism and follows first-order kinetics [9]. Phosphorylation of tyrosine residues was shown to play an important role in the regulation of peanut dual-specificity kinase activity [10]. The role of threonine phosphorylation has not been studied in peanut serine/threonine/tyrosine (STY) protein kinase. However, threonine phosphorylation was shown to play regulatory roles in many protein kinases. Threonine phosphorylation was shown in threonine-glutamate-tyrosine (TEY) motif of activation loop of MAP kinases [11]. Arabidopsis STY protein kinase activity was abolished when threonine in TEY motif was converted to alanine [12].

The tyrosine of TEY motif in peanut dual-specificity kinase was shown to be autoinhibitory [10]. So it is of interest to see the role of threonine in TEY motif in regulating the kinase activity. It is known that residue(s) in the activation loop of kinase domain undergo phosphorylation to regulate kinase activity [13], [14]. Threonine-468 in the activation loop of Arabidopsis somatic embryogenesis receptor kinase-1 was shown to regulate the kinase activity [15]. Threonine-720 of subdomain XI in Catharanthus roseus receptor-like kinase-1 was proposed to be important for both auto- and substrate phosphorylations [16]. There are four threonine residues and a tyrosine residue in the activation loop of peanut STY protein kinase. Involvement of tyrosine residue in the regulation of kinase activity was documented; however, the role of threonine residues is not studied.

In the present study, we show the importance of threonine residues in the regulation of peanut dual-specificity protein kinase activity. This data suggests that peanut STY protein kinase undergoes phosphorylation at multiple sites and also emphasizes the complex regulation of plant dual-specificity kinases.