Archives of Biochemistry and Biophysics 455 (2006) 99–109 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.09.009 Role of threonine residues in the regulation of manganese-dependent arabidopsis serine/threonine/tyrosine protein kinase activity Mamatha M. Reddy, Ram Rajasekharan ¤ Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India Received 10 August 2006, and in revised form 13 September 2006 Available online 4 October 2006 Abstract Tyrosine phosphorylation in plants could be performed only by dual-speciWcity kinases. Arabidopsis thaliana dual-speciWcity protein kinase (AtSTYPK) exhibited strong preference for manganese over magnesium for its kinase activity. The kinase autophosphorylated on serine, threonine and tyrosine residues and phosphorylated myelin basic protein on threonine and tyrosine residues. The AtSTYPK har- bors manganese dependent serine/threonine kinase domain, COG3642. His 248 and Ser 265 on COG3642 are conserved in AtSTYPK and the site-directed mutant, H248A showed loss of serine/threonine kinase activity. The protein kinase activity was abolished when Thr 208 in the TEY motif and Thr 293 of the activation loop were converted to alanine. The conversion of Thr 284 in the activation loop to alanine resulted in an increased phosphorylation. This study reports the Wrst identiWcation of a manganese dependent dual-speciWcity kinase and the importance of Thr 208 , Thr 284 , and Thr 293 residues in the regulation of kinase activity.  2006 Elsevier Inc. All rights reserved. Keywords: Plant dual-speciWcity protein kinase; Regulation of protein kinase activity; Site-directed mutagenesis; MALDI mass spectrometry; Protein phosphorylation Protein phosphorylation is an important and wide- spread biochemical mechanism that regulates many cellular processes [1]. In animal systems, tyrosine phosphorylation controls various signaling pathways that regulate growth and development. Protein tyrosine phos- phatases have been studied in higher plants [2,3]. How- ever, genome-wide analysis of Arabidopsis thaliana predicted one protein tyrosine phosphatase and few dual- speci Wcity phosphatases suggesting that dual-speci Wcity phosphatases predominantly carry out the dephosphoryl- ation [4]. In plants, tyrosine kinase activity was docu- mented in petiole bending in Mimosa pudica [5] and in Catharanthus roseus roots that were transformed by Agro- bacterium rhizogenes [6]. Tyrosine phosphorylation has also been shown to be involved in plant development and embryogenesis [7], during coconut zygotic embryo devel- opment [8] and phytohormone stimulated cell prolifera- tion in Arabidopsis hypocotyls [9]. Pollen-expressed receptor-like kinase 1 of Petunia inXata showed autophos- phorylation on serine and tyrosine suggesting that it may be a dual-speciWcity receptor-like kinase [10]. Although, the presence of tyrosine phosphorylation is well documented in plant systems, the presence of a bonaWde tyrosine kinase has not been established so far. Attempts to clone a tyrosine kinase resulted in the identi W- cation of only dual-speciWcity kinases that phosphorylate on serine, threonine and tyrosine residues [11,12]. Earlier work from our laboratory has shown that the peanut dual-speciWc- ity kinase is stress responsive and developmentally regulated during seed development. This class of dual-speciWcity kinases is shown to undergo intramolecular type of phos- phorylation mechanism and follows Wrst-order kinetics [13]. Genome-wide analysis of Arabidopsis using the delineated tyrosine kinase motifs from animals revealed the presence of only dual-speci Wcity kinases, raising an intriguing possibility that plants lack classical tyrosine kinases [14]. * Corresponding author. Fax: +91 80 23600814. E-mail address: lipid@biochem.iisc.ernet.in (R. Rajasekharan).