Cloning and Characterization of a Novel Serine/Threonine Protein Kinase Expressed in Early Xenopus Embryos

doi:10.1074/jbc.271.24.14430

Cloning and Characterization of a Novel Serine/Threonine Protein Kinase Expressed in Early Xenopus Embryos*

From the Howard Hughes Medical Institute and the Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80262

^§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed:
Howard Hughes Medical Inst., University of Colorado School of Medicine, 4200 East Ninth Ave., P. O. Box C-236, Denver, CO 80262
.

Abstract

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 enopus gg and mbryo inase), 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 (∼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 ∼155 kDa that may be a substrate and/or a regulatory component of the kinase.

Footnotes

↵^‡ Associate of the Howard Hughes Medical Institute.
↵* This work was supported by the Howard Hughes Medical Institute, by National Institutes of Health Grant GM26743, and by a grant to the Molecular Biology Program from the Lucille P. Markey Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U24435[GenBank].
↵¹ The abbreviations used are:

PKA

cAMP-dependent protein kinase

PKI

heat-stable inhibitor of cAMP-dependent protein kinase

PCR

polymerase chain reaction

bp

base pair(s)

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

MES

4-morpholineethanesulfonic acid.
↵² B. E. Kemp, personal communication.
↵³ B. E. Kemp, J. L. Maller, and J.-Y. Su, unpublished results.
- Received January 26, 1996.