Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains

Abstract

Two new kinases of Dietyostelium discoideum were identified by screening of a 2~gtll expression library with a non-receptor kinases, DPYK1 (=SplA) and DPYK2, have phosphotyrosine specific antibody. Amino-acid sequences derived been published by Tan and Spudich [10]. The elimination of from eDNA and genomic clones indicate that DPYK3 is a one of them (SplA) by gene disruption indicates that this protein of 150 kDa and DPYK4, a protein of 75 kDa. The Ckinase is required for viable spore production [11]. In this terminal fragments of each protein were produced in Escherichia paper we describe two new tyrosine kinases of D. discoideum, coli and shown to be autoeatalytically phosphorylated at tyrosine DPYK3 and DPYK4, and the positions of their catalytic conresidues. A common feature of these kinases is the presence of sensus domains in a dendrogram of protein kinases. A chartwo different sequence stretches in tandem that are related to acteristic of both kinases is the presence of two of these dokinase catalytic domains. The sequence relationships of DPYK3 mains, a peculiarity that will be discussed in terms of its and 4 to other protein kinases, and the positions of their catalytic putative phylogenetic origin. domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two 2. Material and methods previously described tyrosine kinases of D. discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu 2.1. Library screening and DNA sequencing strictu. Domain II in DPYK4 is found on a different branch close A ~,gtl 1-cDNA expression library of D. discoideum AX3, purchased to serinelthreonine kinases, from Clontech, was screened with iodinated anti-phosphotyrosine mAb 5E2 [12]. The cDNA inserts of phages from 11 plaques recogKey words." Dictyostelium; Dendrogram; Protein tyrosine nized by mAb 5E2 were amplified by PCR with ~.gt 11 specific primers, subcloned into pUC19 and sequenced. Based on cDNA and genomic kinase DNA, the DPYK3 and DPYK4 sequences were completed using sequence specific PCR, inverted PCR, and enrichment mediated PCR approaches [13,14]. The continuity of the obtained sequence fragments was confirmed by the sequencing of two overlapping PCR products of 1. Introduction genomic DNA of D. discoideum AX2 covering the entire DPYK3 coding region including one intron and by the sequencing of one During development of the eukaryotic microorganism Diccontinuous PCR product of genomic DNA comprising the entire tyostelium discoideum, cell interactions are mediated by a ser-DPYK4 coding region plus two introns.ies of chemical signals [1]. These include cAMP and folic acid 2.2. Expression in E. coli that act as chemoattractants, and as ligands in signal trans-A [3-Gal/DPYK3 fusion protein containing amino acids 918-1338 duction pathways controlling gene activities in development, of DPYK3 encoded by the original ~gtl 1-cDNA clone was expressed in E. coli Y1089. C...

An SH2-domain-containing kinase negatively regulates the phosphatidylinositol-3 kinase pathway

“…To date, we have not found an SHK1 homolog in the databases of other organisms. However, this is not unexpected, because Dictyostelium protein kinases capable of phosphorylating on tyrosine residues, even those that are monospecific tyrosine kinases, tend not to be closely related to metazoan kinases (Jungbluth et al 1995;Adler et al 1996;Nuckolls et al 1996;Kim et al 1999). We found that the SHK1 SH2 domain is most closely related to that of the p110␣ PI3K adaptor p55.…”

Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling

“…VSK3 was identified bioinformatically and is a transmembrane kinase with a role in phagocytosis (20). The other kinases were identified by expressing cDNA libraries in Escherichia coli and monitoring phosphotyrosine production in individual bacterial colonies using an antibody (21)(22)(23). Zak1 and Zak2 (originally termed "Pyk4") phosphorylate GSK3 on tyrosine residues, increasing its activity (23)(24)(25).…”

Jak-Stat signal transduction pathway through the eyes of cytokine class II receptor complexes

“…Numerous divergent interacting partners of Jaks and Stats have been discovered revealing or just suggesting many new, unpredictable functions for Jaks and Stats (KlingmuÈ ller et al, 1995;Marrero et al, 1995;Yamauchi et al, 1997;Guillet-Deniau et al, 1997;Pollack et al, 1999Pollack et al, , 2000. The Jak-Stat signal transduction pathway has evolved, likely together with cytokine families, from such lower eukaryotes as Dictyostelium discoideum (Adler et al, 1996;Kawata et al, 1997;Araki et al, 1998). Along the evolutionary path, it acquired many new alternative functions but also retained at least partially, some original ancestral functions.…”