Casein Kinase II Catalyzes Tyrosine Phosphorylation of the Yeast Nucleolar Immunophilin Fpr3*
- From the Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3204
Abstract
In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1. In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr. Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII). Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII. Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII. Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature. Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII. Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186. These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo andin vitro and thus possesses dual specificity. These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM44173 (to G. S. M.) and GM21841 (to J. T.), and by the facilities of the University of California Cancer Research Laboratory.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.
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↵‡ Present address: Charybdis Corp., 22021 20th Avenue S.E., Bothell, WA 98021.
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↵§ To whom correspondence should be addressed: Dept. of Molecular and Cell Biology, Div. of Biochemistry and Molecular Biology, University of California at Berkeley, 401 Barker Hall #3204, Berkeley, CA 94729-3204. Tel.: 510-642-1508; Fax: 510-642-7000; E-mail:smartin{at}socrates.berkeley.edu.
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↵1 L. Wilson, unpublished results.
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↵2 The abbreviations used are: MEK, mitogen-activated protein kinase kinase; CKII, casein kinase II; CKI, casein kinase I; yCKII, yeast CKII; hCKII, human CKII; dCKII,Drosophila CKII; GST, glutathione S-transferase; Ptp1, protein-tyrosine phosphatase 1; PAGE, polyacrylamide gel electrophoresis.
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- Received January 7, 1997.
- Revision received March 13, 1997.
