Cloning and characterization of a novel serine/threonine protein kinase expressed in early Xenopus embryos.
We have cloned from a Xenopus ovary cDNA library a novel protein kinase gene whose expression peaks in the oocyte and unfertilized egg, begins to decrease gradually after fertilization, and disappears during the gastrulation stage of embryogenesis. The cloned gene, termed XEEK1 (for Xenopus egg and embryo kinase), encodes a protein with a predicted molecular mass of 49 kDa. Bacterially expressed XEEK1 migrates at 57 kDa upon polyacrylamide gel electrophoresis analysis, and a XEEK1-specific antibody recognizes a protein of 57 kDa in Xenopus oocyte and egg extracts. The XEEK1 kinase domain shares 35% identity (approximately 65% similarity) with the yeast SNF1 kinase and related kinases. However, expression of XEEK1 does not complement a snf1 deletion mutation in yeast, which suggests that it is probably not a Xenopus homolog of SNF1. Recombinant XEEK1 protein autophosphorylates on threonine residues in vitro in a reaction that prefers Mn2+ to Mg2+ ions. Site-directed mutagenesis of the conserved lysine residue (Lys-81) within the kinase domain to isoleucine totally abolishes kinase activity, and threonine 192 has been identified as the autophosphorylation site. This site is distinct from the conserved threonine (Thr-215 in XEEK1) present in the protein kinase activation loop that is the site of autophosphorylation for many protein kinases. XEEK1 is a substrate for the cyclic AMP-dependent protein kinase both in vitro and in vivo, suggesting a possible mode of regulation of XEEK1. An immunoprecipitate of oocyte/egg extracts with anti-XEEK1 serum contains a protein of approximately 155 kDa that may be a substrate and/or a regulatory component of the kinase.
Su
J Y
JY
Howard Hughes Medical Institute and the Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262, USA.
Erikson
E
E
Maller
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000201:enzymology*; D005260:Female; D005306:Fertilization; D018507:Gene Expression Regulation, Developmental; D008297:Male; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000196:embryology
D017931:DNA Primers; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C100358:STK11 protein, Xenopus; D017346:Protein Serine-Threonine Kinases