Identification of the major Mr 100,000 substrate for calmodulin-dependent protein kinase III in mammalian cells as elongation factor-2.
The major substrate for Ca2+/calmodulin-dependent protein kinase III in mammalian cells is a species of Mr 100,000 that has a primarily cytoplasmic localization. This substrate has now been identified as elongation factor-2 (EF-2), a protein that catalyzes the translocation of peptidyl-tRNA on the ribosome. The amino acid sequence of 18 residues from the N-terminal of the Mr 100,000 CaM-dependent protein kinase III substrate purified from rat pancreas was found to be identical to the N-terminal sequence of authentic rat EF-2 as previously deduced from nucleic acid sequencing of a cDNA (Kohno, K., Uchida, T., Ohkubo, H., Nakanishi, S., Nakanishi, T., Fukui, T., Ohtsuka, E., Ikehara, M., and Okada, Y. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4978-4982). CaM-dependent protein kinase III phosphorylated EF-2 in vitro with a stoichiometry of approximately 1 mol/mol on a threonine residue. Amino acid sequencing of the purified tryptic phosphopeptide revealed that this threonine residue lies within the sequence: Ala-Gly-Glu-Thr-Arg-Phe-Thr-Asp-Thr-Arg (residues 51-60 of EF-2). The Mr 100,000 protein was stoichiometrically ADP-ribosylated in vitro by the addition of diphtheria toxin and NAD. The Mr 100,000 protein was photoaffinity labeled with a GTP analog and the protein had an endogenous GTPase activity that could be stimulated by the addition of salt-washed ribosomes. These properties are all characteristic of EF-2. Dephospho-EF-2 could support poly(U)-directed polyphenylalanine synthesis in a reconstituted elongation system when combined with EF-1. In the same system, phospho-EF-2 was virtually inactive in supporting polypeptide synthesis; this effect could be reversed by dephosphorylation of phospho-EF-2. These results suggest that intracellular Ca2+ inhibits protein synthesis in mammalian cells via CaM-dependent protein kinase III-catalyzed phosphorylation of EF-2.
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
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Palfrey
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008970:Molecular Weight; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D000596:Amino Acids; D002147:Calmodulin; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D000246:Adenosine Diphosphate Ribose; D006160:Guanosine Triphosphate; D011494:Protein Kinases