Casein Kinase II Catalyzes Tyrosine Phosphorylation of the Yeast Nucleolar Immunophilin Fpr3
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.12961
ISSN1083-351X
AutoresLinda K. Wilson, Namrita Dhillon, Jeremy Thorner, Greg S. Martin,
Tópico(s)Peptidase Inhibition and Analysis
ResumoIn 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. 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. Protein kinases with dual specificity are able to phosphorylate either themselves and/or their target substrates at tyrosine and at serine or threonine (1Lindberg R.A. Quinn A.M. Hunter T. Trends Biochem. 1992; 17: 114-119Abstract Full Text PDF PubMed Scopus (195) Google Scholar). A number of protein kinases with dual specificity have been identified in budding yeast (Saccharomyces cerevisiae) and in fission yeast (Schizosaccharomyces pombe). These enzymes are likely to be responsible for all of the protein-bound phosphotyrosine found in these organisms, since no tyrosine-specific protein kinases have yet been detected in either yeast. Examples of such protein kinases in S. cerevisiae include: the mitogen-activated protein kinase kinase (MEK)2 homologs Ste7 (2Errede B. Levin D.E. Curr. Opin. Cell Biol. 1993; 5: 254-260Crossref PubMed Scopus (187) Google Scholar), Pbs2 (3Brewster J.L. deValoir T. Dwyer N.D. Winter E. Gustin M.C. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1032) Google Scholar), Mkk1 and Mkk2 (4Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (259) Google Scholar); a Wee1 homolog, Swe1 (5Booher R.N. Deshaies R.J. Kirschner M.W. EMBO J. 1993; 12: 3417-3426Crossref PubMed Scopus (288) Google Scholar); the glycogen synthase kinase-3 homolog, Mck1 (6Lim M.-Y. Dailey D. Martin G.S. Thorner J. J. Biol. Chem. 1993; 268: 21155-21164Abstract Full Text PDF PubMed Google Scholar); and a protein kinase involved in DNA damage checkpoint control, Spk1/Rad53 (7Sun Z. Fay D.S. Marini F. Foiani M. Stern D.F. Genes Dev. 1996; 10: 395-406Crossref PubMed Scopus (273) Google Scholar). With one exception, all of the known phosphotyrosine-containing proteins in S. cerevisiae are themselves protein kinases, for example, the cell cycle kinase Cdc28 (8Sorger P.K. Murray A.W. Nature. 1992; 355: 365-368Crossref PubMed Scopus (216) Google Scholar) and the mitogen-activated protein kinases, Kss1 (9Ma D. Cook J.G. Thorner J. Mol. Biol. Cell. 1995; 6: 889-909Crossref PubMed Scopus (68) Google Scholar), and Fus3 (10Gartner A. Nasmyth K. Ammerer G. Genes Dev. 1992; 6: 1280-1292Crossref PubMed Scopus (221) Google Scholar). The exception is Fpr3, which is an abundant nucleolar protein that is a member of the FK506-binding subfamily of immunophilins with an as yet unknown physiological function (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar). We have shown previously that phosphotyrosyl-Fpr3 is a cellular substrate of the S. cerevisiae protein-tyrosine phosphatase, Ptp1. This phosphatase directly dephosphorylates phosphotyrosyl-Fpr3 in vitro, and prevents accumulation of phosphotyrosyl-Fpr3 in vivo(12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The phospho-Tyr that appears in Fpr3 in Ptp1-deficient cells is unlikely to result from autophosphorylation, because Fpr3 lacks homology to any known protein kinase and does not display detectable protein kinase activity in vitro. Hence, we sought to identify the protein kinase responsible for phosphorylating Fpr3 at Tyr. The COOH-terminal third of Fpr3 comprises its FK506-binding and peptidyl-prolyl-cis,trans-isomerase domain, whereas the NH2-terminal two-thirds of Fpr3 has homology to other nucleolar proteins, like nucleolin, and is responsible for localization of Fpr3 to the nucleolus (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar). The NH2-terminal segment is phosphorylated at Ser, Thr, and Tyr, and in this region many of the potential phosphoacceptor residues are flanked by acidic residues. This arrangement suggested that Fpr3 might be a substrate for casein kinase II (CKII), 2The 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. because this enzyme has a requirement for acidic substrates (13Tuazon T.P. Traugh J.A. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 123-165PubMed Google Scholar) and because it is also localized to the nucleolus (14Pfaff M. Anderer F.A. Biochim. Biophys. Acta. 1988; 969: 100-109Crossref PubMed Scopus (53) Google Scholar). Furthermore, we previously demonstrated that the exclusive site of tyrosine phosphorylation within Fpr3 is Tyr184 (underlined), which also resides in a highly acidic sequence (DEDEDADIYDSEDYDLT) within the NH2-terminal domain (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). This observation raised the further possibility that phosphorylation of Fpr3 at Tyr might be catalyzed by CKII. CKII is a well characterized, Ser/Thr-directed protein kinase with a broad range of reported substrates in vivo and with multiple roles in signal transduction (15Allende J.W. Allende C.C. FASEB J. 1995; 9: 313-323Crossref PubMed Scopus (588) Google Scholar, 16Litchfield D.W. Luscher B. Mol. Cell. Biochem. 1993; 127/128: 187-199Crossref Scopus (162) Google Scholar). CKII is composed of catalytic (α and/or α′) and regulatory (β and/or β′) subunits, associated as either an α2β2 or an αα′ββ′ tetramer. In S. cerevisiae, the α and α′ subunits of CKII are encoded by the CKA1 and CKA2 genes, respectively (17Padmanabha R. Chen-Wu J.L.-P. Hanna D.E. Glover C.V.C. Mol. Cell. Biol. 1990; 10: 4089-4099Crossref PubMed Scopus (306) Google Scholar). Disruption of either gene still permits cell growth, whereas disruption of both genes is lethal. Yeast mutants which produce temperature-sensitive CKII undergo growth arrest within the time of about one cell cycle after shift to restrictive temperature (18Hanna D.E. Rethinaswamy A. Glover C.V.C. J. Biol. Chem. 1995; 270: 25905-25914Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Half of the arrested cells are unbudded, and the other half have large buds, suggesting that CKII activity is required for progression through both G1 and G2/M. Examination of natural protein substrates, and systematic studies using synthetic peptide substrates, indicate that the consensus sequence for CKII phosphorylation is Ser/Thr-X-X-Neg (where Neg indicates a negatively-charged residue or a residue carrying a negatively-charged modification), although acidic residues at positions spanning from −2 to +7 can act as positive specificity determinants for CKII, and an acidic residue at position +3 is not an absolute requirement (19Meggio F. Marin O. Pinna L.A. Cell. Mol. Biol. Res. 1994; 40: 401-409PubMed Google Scholar). The order of efficacy of negatively-charged specificity determinants at the +3 position is thought to be: P-Tyr > P-Ser > Glu > P-Thr. Thus, phosphorylated Tyr and phosphorylated Ser surpass Glu, providing biochemical support for the possibility that CKII may participate in ordered multisite phosphorylation (20Meggio F. Perich J.W. Reynolds E.C. Pinna L.A. FEBS Lett. 1991; 279: 307-309Crossref PubMed Scopus (24) Google Scholar). Here we demonstrate that phosphorylation of Fpr3 at Tyr, as well as at Ser and Thr, is dependent on CKII activity in vivo. Furthermore, we present evidence that CKII phosphorylates Tyr184 in vitro by a two-step mechanism involving prior phosphorylation of the Ser residue in the +2 position. These results indicate that CKII is not exclusively a Ser/Thr-directed protein kinase, but rather has dual specificity. Moreover, our results indicate that CKII has the previously unrecognized capacity to use P-Ser as a specificity determinant to permit subsequent phosphorylation of a nearby Tyr. Standard methods were used for growth and transformation of yeast (21Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., Struhl, K., Current Protocols in Molecular Biology, 1994, Wiley-Interscience, New York.Google Scholar). Strains YDH6 and YDH8 (generous gift of C. Glover, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA) harbor disruptions of both CKA1 andCKA2; in strain YDH6, the CKII deficiency is complemented by a wild-type copy of CKA2, whereas in strain YDH8 the deficiency is complemented by a temperature-sensitive allele (cka2-8) carried on a CEN plasmid (18Hanna D.E. Rethinaswamy A. Glover C.V.C. J. Biol. Chem. 1995; 270: 25905-25914Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). To generate strains YDH6200 and YDH8200, the PTP1 gene in strains YDH6 and YDH8 was disrupted by homologous recombination using a 1.25-kilobase pair PvuII-AseI fragment from the plasmid pGEM-ptp1::URA3 (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To overexpress Drosophila CKII in yeast, the PTP1strain PJ55–16A and the otherwise isogenic ptp1Δ strain PJ55–16C (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) were transformed with the CEN plasmid pDH5 (a generous gift of C. Glover), which contains cDNAs encoding the α and β subunits of Drosophila CKII, each expressed divergently under control of the dual GAL1/10 promoter (17Padmanabha R. Chen-Wu J.L.-P. Hanna D.E. Glover C.V.C. Mol. Cell. Biol. 1990; 10: 4089-4099Crossref PubMed Scopus (306) Google Scholar). Residues 20–290 from the noncatalytic NH2-terminal region of Fpr3 (Fpr3N) were fused in-frame to the COOH terminus of glutathione S-transferase (GST) yielding GST-Fpr3N. GST-Fpr3N(Y184F) is an otherwise identical fusion protein in which the tyrosine phosphoacceptor site was mutated to Phe. The GST fusions were expressed in bacteria and purified as described previously (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). GST-Fpr3N (bound to glutathione-Sepharose beads, Pharmacia) or soluble Fpr3N (generated by thrombin cleavage of GST-Fpr3N), were used as kinase substrates. Purified S. cerevisiae CKII α′αβ′β heterotetramer (yCKII) (generous gift of C. Glover), was purified to greater than 95% homogeneity by a procedure described elsewhere (22Bidwai A.P. Reed J.C. Glover C.V. Arch. Biochem. Biophys. 1994; 309: 348-355Crossref PubMed Scopus (40) Google Scholar). Human CKII α2β2 tetramer (hCKII), purified fromEscherichia coli expressing both α and β subunits, was purchased from New England Biolabs, Beverly, MA. Unless otherwise noted, phosphorylation reactions were carried out in reaction mixtures (20 μl final volume) containing 1 μg of Fpr3 substrate, 30 ng of CKII, 200 μm ATP, 20 mm Tris-HCl (pH 7.2), 50 mm KCl, and 10 mm MgCl2. In some reactions, [γ32-P]ATP was included at the specific activities indicated in the figure legends. Reactions were carried out at 30 °C for the times indicated, and terminated by addition of one-tenth volume of 0.5 m EDTA. Reaction products were resolved by SDS-PAGE. When yeast lysates were used as the source of protein kinase, reactions were carried out as described previously (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Briefly, YDH6 or YDH8 cells were broken by glass bead lysis, and the lysates were clarified by centrifugation. Glutathione-Sepharose beads bearing GST-Fpr3N were incubated with the lysates at 30 °C, then rinsed briefly and incubated in the same buffer used for CKII reactions containing either 100 μm non-radiolabeled ATP or 100 μm[γ-32P]ATP (specific activity 5 Ci/mmol), as indicated in the figure legends. To generate GST-Fpr3N phosphorylated at serine and threonine, but not at tyrosine, GST-Fpr3N bound to glutathione-Sepharose beads was incubated with CKII and unlabeled ATP for 1 h as described above. The beads were washed 3 times with 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 20 mm EDTA, resuspended in the same solution and incubated for 30 min at 30 °C with 50 μg/ml bacterially-expressed ScPtp1 (which was prepared as described previously (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar)). The beads were then washed 3 times in kinase reaction buffer containing 100 μm Na3V04prior to their use in CKII reactions (see Fig. 5 B). Peptides were synthesized by D. King (HHMI, University of California, Berkeley, CA) using Fmoc chemistry on an automated ABI synthesizer (Model 431-A, Applied Biosystems, Foster City, CA). The composition of the peptides was assessed by electrospray ionization mass spectroscopy, and purity was always greater than 95%. Peptides corresponding to residues 178–191 of Fpr3 (DEDADIYDSEDYDL) were prepared, in which phosphoserine, glutamic acid, or alanine were substituted for Ser186 (underlined). Peptide phosphorylation reactions (45 μl final volume) contained 1 mg/ml peptide, 1.5 μg/ml human CKII, 100 μm[γ32-P]ATP (5 Ci/mmol), 20 mm Tris-HCl (pH 7.2), 50 mm KCl, and 10 mm MgCl2. Samples (5 μl) were removed at intervals and mixed with 2 μl of 0.5m EDTA to terminate the reaction. Reaction products were analyzed by spotting 3 μl of each sample onto 2-cm squares of DEAE-cellulose paper (NA45, Schleicher & Schuell, Keene, NH) and washing the paper 4 times for 5 min each in 50 mm Tris-HCl (pH 7.4), 100 mm NaCl. The papers were then rinsed in acetone and dried and the amount of radiolabel incorporated determined by counting in a liquid scintillation spectrometer. Strains YDH6200 and YDH8200 were grown to stationary phase (A 600 nm = 4) and lysed by vigorous agitation with glass beads as described previously (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar) in lysis buffer (100 mm NaCl, 50 mm Tris-HCl (pH 7.2), 5 mm EDTA, 0.1% Triton X-100, 12 μm benzamidine, 5 μmphenanthroline, 30 μm phenylmethylsulfonyl fluoride, 100 μm Na3V04, and 0.5 μg/ml of each of the following protease inhibitors: antipain, leupeptin, chymostatin, aprotinin, and pepstatin). Lysates were clarified by centrifugation for 15 min at 10,000 × g, and the resulting supernatant fractions were centrifuged at 100,000 ×g for 40 min. Proteins were eluted from the pellet by stirring the resuspended particulate material for 1 h at 4 °C in 0.5 ml of lysis buffer containing a final concentration of 1m NaCl. Insoluble particulate matter was removed from the suspension by centrifugation at 100,000 × g. The resulting supernatant extract was diluted 6-fold in lysis buffer and incubated at 4 °C for 16 h with agarose beads to which had been coupled immunoglobulin from rabbits immunized against the amino terminus of Fpr3 (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar). The beads were then washed 4 times in lysis buffer and solubilized by boiling in SDS-PAGE sample buffer. The immunoprecipitated proteins were resolved by SDS-PAGE and subjected to immunoblot analysis as described below. Protein kinase reaction mixtures, samples of yeast lysates (100 μg), or solubilized immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting as described previously (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The following antibodies (1 μg of protein/ml) were used as probes: anti-phosphotyrosine mAb 4G10 (23Drucker B.J. Mamon H.J. Roberts T.M. N. Engl. J. Med. 1989; 321: 1383-1391Crossref PubMed Scopus (245) Google Scholar) (Upstate Biotechnology, Lake Placid, NY), immunoglobulin from rabbits immunized against the amino terminus of Fpr3 (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar), or serum raised against Drosophila CKII holoenzyme (24Dahmus G.K. Glover C.V.C. Brutlag D.L. Dahmus C.E. J. Biol. Chem. 1984; 259: 9001-9006Abstract Full Text PDF PubMed Google Scholar) (generous gift of C. Glover). Phosphoamino acid analyses were carried out as described previously (25Boyle W.J. Geer P.V.D. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). Briefly, proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore). Following autoradiography, the bands corresponding to phosphorylated Fpr3N were excised and subjected to partial acid hydrolysis. After addition of P-Tyr, P-Thr, and P-Ser, which served as both carriers and internal standards, the dried samples were resolved electrophoretically on thin-layer cellulose plates (Merck) at pH 1.9 in the first dimension and at pH 3.7 in the second dimension. Radiolabeled phosphoamino acids detected by autoradiography and unlabeled phosphoamino acids were detected by staining with ninhydrin. We have shown previously that yeast lysates contain an activity capable of binding to Fpr3 and of phosphorylating it in vitro at Ser, Thr, and Tyr (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The resulting relative phosphoamino acid content is similar to that found in Fpr3 phosphorylated in vivo (phosphoserine, 85%; phosphothreonine, 11%; and phosphotyrosine, 4%) (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). To determine whether CKII is required for this phosphorylation, strain YDH6, acka1Δ cka2Δ strain in which the CKII deficiency is complemented by wild-type CKA2, and strain YDH8, an isogenic strain in which the CKII deficiency is complemented by a temperature-sensitive allele (cka2–8) (18Hanna D.E. Rethinaswamy A. Glover C.V.C. J. Biol. Chem. 1995; 270: 25905-25914Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), were grown at the permissive temperature (25 °C), and cell lysates were prepared. These lysates were incubated with glutathione-Sepharose beads loaded with GST-Fpr3N, a glutathione S-transferase fusion protein containing the NH2-terminal portion of Fpr3 (see "Experimental Procedures"). The beads were washed and incubated at 30 °C in a reaction buffer containing [γ32-P]ATP, and the reaction products analyzed by SDS-PAGE and autoradiography. Lysate from cells expressing wild-type CKA2 mediated efficient phosphorylation of GST-FprN (Fig. 1 A, Lane 1), whereas lysate from cells expressing thecka2–8 temperature-sensitive mutant did not (Fig. 1 A, Lane 2). These results indicate that the activity in yeast lysates that phosphorylates Fpr3 at serine, threonine, and tyrosine residues is CKII-dependent. YDH8 cells exhibit phenotypic changes characteristic of CKII depletion even at the permissive temperature and fail to grow at temperatures above 33 °C (18Hanna D.E. Rethinaswamy A. Glover C.V.C. J. Biol. Chem. 1995; 270: 25905-25914Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Thus, the inability of lysates of YDH8 cells grown at the permissive temperature to phosphorylate Fpr3 probably reflects inactivation of the mutant CKII during incubation at 30 °C in vitro. To examine the effect of CKII deficiency on phosphorylation of Fpr3 at tyrosine, kinase reactions similar to those described above were carried out using non-radioactive ATP, and the reaction products were examined by immunoblotting with anti-phosphotyrosine antibody (Fig. 1 B). Incubation of GST-Fpr3N with a lysate of YDH6 cells resulted in tyrosine phosphorylation of GST-Fpr3N in the subsequent kinase reaction (Fig. 1 B, Lane 1), whereas incubation with a lysate of the CKII-deficient YDH8 cells (Fig.1 B, Lane 2) or with buffer alone (Fig. 1 B, Lane 4) did not. In addition, tyrosine phosphorylation was not detected when the Y184F mutant of GST-Fpr3N (GST-Fpr3NY184F) was used as kinase substrate with the active lysate from YDH6 cells (Fig. 1 B, Lane 3). Therefore, the kinase responsible for the tyrosine phosphorylation of Fpr3 in yeast lysates requires both the function of CKII and the integrity of the Tyr site in Fpr3 that is known to be phosphorylated in vivo. To enhance detection of phosphotyrosine in Fpr3 in vivo, derivatives of YDH6 and YDH8 lacking the PTP1gene were generated by gene disruption as described previously (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The resulting strains, YDH6200 and YDH8200, were grown to mid-exponential phase at the permissive temperature (25 °C). Cell lysates were subjected to immunoprecipitation with anti-Fpr3 antibody. Both lysates and immunoprecipitates were resolved by SDS-PAGE and the phosphotyrosyl proteins present were examined by immunoblotting with anti-phosphotyrosine antibody. Phosphotyrosyl-Fpr3 was detected in the lysate of YDH6200 cells (Fig. 2 A, Lane 1), but not in the lysate of YDH8200 (Fig. 2 A, Lane 2), even though immunoblot analysis of the same lysates using anti-Fpr3 antibody demonstrated that the total amount of Fpr3 protein was essentially the same in both extracts (Fig. 2 A, Lanes 3 and 4). Likewise, Fpr3 immunoprecipitated from YDH6200 was phosphorylated at tyrosine, while Fpr3 immunoprecipitated from YDH8200 was not (Fig.2 B). These results indicate that, as in vitro, tyrosine phosphorylation of Fpr3 in vivo is also CKII-dependent. It is interesting to note that an unidentified protein (∼50 kDa) also appeared to be phosphorylated at tyrosine in a CKII-dependent manner (Fig. 2, Lane 1); this protein did not appear to be derived from Fpr3 because it did not react with anti-Fpr3 antibody. In contrast to budding yeast CKII (an αα′ββ′ heterotetramer encoded by four separate genes), Drosophila CKII (dCKII) is an ααββ tetramer and is encoded by only two genes, facilitating ectopic expression; moreover expression of dCKII can complement yeast cells lacking endogenous CKII (17Padmanabha R. Chen-Wu J.L.-P. Hanna D.E. Glover C.V.C. Mol. Cell. Biol. 1990; 10: 4089-4099Crossref PubMed Scopus (306) Google Scholar). To determine if high-level expression of dCKII could affect the extent of tyrosine phosphorylation in Fpr3, a plasmid encoding the α and β subunits of dCKII under dual control of the divergent GAL1/10 promotor was introduced into ptp1 and PTP1 strains. The resulting strains were grown in either galactose medium to induce expression of dCKII, or in glucose medium to repress dCKII expression. Gal-dependent production of dCKII was verified by immunoblot analysis of cell lysates with an antibody specific for dCKII (Fig. 3, lower panel). Immunoblot analysis using anti-Fpr3 antibodies indicated that the abundance of Fpr3 was not affected by expression of dCKII (data not shown). However, expression of dCKII resulted in enhanced tyrosine phosphorylation of Fpr3 in theptp1 strain as detected by an increase in the signal obtained with the anti-phosphotyrosine antibody (Fig. 3, upper panel, Lane 2). This increase was not observed in thePTP1 strain (Fig. 3, upper panel, Lane 3), indicating that the endogenous level of Ptp1 was sufficient to block the hyperphosphorylation of Fpr3 that resulted from dCKII expression. The results described above suggest that CKII either directly phosphorylates Fpr3, or activates some other protein kinase which in turn phosphorylates Fpr3 at tyrosine. To determine whether CKII can directly phosphorylate Fpr3 at Tyr184, CKII heterotetramer purified to greater than 95% from S. cerevisiae (yCKII) was incubated with [γ-32P]ATP and, as substrate, either GST-Fpr3N or GST-Fpr3N(Y184F). The reaction products were resolved by SDS-PAGE and the phosphorylated fusion proteins were excised and subjected to phosphoamino acid analysis. yCKII phosphorylated Fpr3 at serine, threonine, and tyrosine (Fig.4 A, Box 1); the Fpr3 phosphorylated in vitro had a phosphoamino acid composition similar to that of Fpr3 phosphorylated in vivo (12Wilson L.K. Benton B.M. Zhou S. Thorner J. Martin G.S. J. Biol. Chem. 1995; 270: 25185-25193Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). GST-Fpr3N(Y418F) was not phosphorylated at tyrosine, confirming that Tyr184 is the site of tyrosine phosphorylation (Panel A, Box 2). In further support of the conclusion that CKII itself, and not a contaminating kinase, was responsible for the tyrosine phosphorylation of Fpr3N, we found that human CKII (hCKII), expressed and purified fromE. coli, also phosphorylated Fpr3 at Tyr184: phosphotyrosine was detected both by phosphoamino acid analysis of radiolabeled Fpr3N (Fig. 4, Panel A, Box 3) and by anti-phosphotyrosine immunoblotting of Fpr3N phosphorylated by hCKII in the presence of non-radiolabeled ATP (Fig. 4, Panel B). Autophosphorylation of CKII at Tyr was not detected. Because E. coli lacks detectable protein-tyrosine kinase activity (26Wang J.Y.J. Baltimore D. J. Biol. Chem. 1985; 260: 64-71Abstract Full Text PDF PubMed Google Scholar, 27Foster R.S. Thorner J. Martin G.S. J. Bacteriol. 1989; 171: 272-279Crossref PubMed Scopus (35) Google Scholar), these results provide strong evidence that CKII can directly phosphorylate Fpr3 at Tyr184 and hence that CKII is a protein kinase with dual specificity. To explore the mechanism of Tyr184 phosphorylation by CKII, we examined the kinetics of serine, threonine, and tyrosine phosphorylation of Fpr3 in vitro. hCKII was incubated with Fpr3N and [γ-32P]ATP, and the phosphoamino acid composition of Fpr3N was determined at various times during the reaction. Tyrosine phosphorylation occurred at a slower rate than either Ser or Thr phosphorylation and appeared biphasic (Fig.5 A). Tyrosine phosphorylation of Fpr3N reached a stoichiometry of 0.2 mol
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