Casein Kinase II Catalyzes Tyrosine Phosphorylation of the Yeast Nucleolar Immunophilin Fpr3

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. 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 D.E. A. Glover C.V.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). of the cells are and the other have that CKII activity is for both and of protein and using synthetic peptide indicate that the sequence for CKII phosphorylation is a negatively-charged or a carrying a negatively-charged acidic residues at from to as specificity for CKII, and an acidic at position is not an requirement F. Cell. Mol. Biol. Res. 1994; Google Scholar). The of of negatively-charged specificity at the position is to P-Ser phosphorylated Tyr and phosphorylated Ser for the possibility that CKII in phosphorylation F. J.W. 1991; PubMed Scopus (24) Google Scholar). we that phosphorylation of Fpr3 at Tyr, as well as at Ser and Thr, is dependent on CKII activity in Furthermore, we that CKII phosphorylates Tyr184 in vitro by a two-step phosphorylation of the Ser in the +2 These results indicate that CKII is not a Ser/Thr-directed protein but has dual specificity. Moreover, results indicate that CKII has the previously to P-Ser as a specificity determinant to subsequent phosphorylation of a Tyr. were used for growth and of yeast F. R. D. J. J. in Scholar). and of of and of of both CKA1 in the CKII is by a of whereas in the is by a temperature-sensitive allele on a D.E. A. Glover C.V.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). and the gene in and was by using a from the (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). CKII in the and the (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 with the of which contains the α and subunits of CKII, of the dual (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). from the NH2-terminal region of Fpr3 were to the of glutathione is an protein in which the tyrosine phosphoacceptor site was to The were in and purified as 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). to or by of were used as kinase S. cerevisiae CKII of was purified to than by a Glover Biochem. Biophys. 1994; PubMed Scopus Google Scholar). CKII α2β2 purified both α and subunits, was from phosphorylation were in containing of Fpr3 of CKII, and In was at the in the were at for the and by of of were by yeast lysates were used as the of protein were as 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). or cells were by and the lysates were by were with the lysates at and in the used for CKII containing either or activity as in the phosphorylated at serine and but not at to was with CKII and for as The were with in the and for at with bacterially-expressed was as 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 The were in kinase containing to their in CKII were by D. of using on an The of the peptides was by and was than corresponding to residues of Fpr3 were in which or were for Ser186 phosphorylation contained human CKII, and were at and with of to the were by of of and the for in The were in and and the of by in a and were grown to and by with as previously (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar) in and of of the and were by for at and the were at for were from the by the for at in of containing a of was from the by at The was in and at for with to which been from the of Fpr3 (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar). The were in and by in The proteins were by and to as Protein kinase of yeast lysates or were by and to as 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 of were used as J. PubMed Scopus Google Scholar) from the of Fpr3 (11Benton B. Zang J.-H. Thorner J. J. Cell Biol. 1994; 127: 623-639Crossref PubMed Scopus (76) Google Scholar), or raised CKII Glover C.V.C. J. Biol. Chem. 259: Full Text PDF PubMed Google Scholar) of were as previously Hunter T. 1991; PubMed Scopus Google Scholar). proteins were by and to the corresponding to phosphorylated were and to of and which as both and the were on at in the and at in the detected by and were detected by with We have shown previously that yeast lysates contain an activity of 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 is to that found in Fpr3 phosphorylated in vivo and (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). CKII is for this in which the CKII is by and an in which the CKII is by a temperature-sensitive allele D.E. A. Glover C.V.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), were grown at the permissive temperature and cell lysates were These lysates were with with a glutathione protein containing the NH2-terminal of Fpr3 The were and at in a containing and the by and from cells CKA2 efficient phosphorylation of whereas from cells temperature-sensitive not These results indicate that the activity in yeast lysates that phosphorylates Fpr3 at and tyrosine residues is cells of CKII even at the permissive temperature and to at D.E. A. Glover C.V.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). the of lysates of cells grown at the permissive temperature to phosphorylate Fpr3 of the CKII at in vitro. the of CKII on phosphorylation of Fpr3 at kinase to were using and the were by with of with a of cells in tyrosine phosphorylation of in the subsequent kinase whereas with a of the cells or with In tyrosine phosphorylation was not detected when the of was used as kinase substrate with the from cells the kinase responsible for the tyrosine phosphorylation of Fpr3 in yeast lysates both the function of CKII and the of the Tyr site in Fpr3 that is known to be phosphorylated in of phosphotyrosine in Fpr3 in of and the were by gene disruption as 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 and were grown to at the permissive temperature Cell lysates were to with lysates and were by and the proteins were by with was detected in the of cells but not in the of even of the lysates using demonstrated that the of Fpr3 protein was the in both and Likewise, Fpr3 from was phosphorylated at Fpr3 from was not These results indicate as in vitro, tyrosine phosphorylation of Fpr3 in vivo is also is to that an protein also to be phosphorylated at tyrosine in a this protein not to be from Fpr3 because it not with In to budding yeast CKII αα′ββ′ encoded by CKII is an and is encoded by only genes, expression of yeast cells 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). expression of the of tyrosine phosphorylation in Fpr3, a the α and subunits of dual of the was and The were grown in either to expression of or in to of was by of cell lysates with an for using that the of Fpr3 was not by expression of not expression of in enhanced tyrosine phosphorylation of Fpr3 in as detected by an in the signal with the This was not in that the endogenous of was to the of Fpr3 that from The results that CKII either directly phosphorylates Fpr3, or other protein kinase which in phosphorylates Fpr3 at CKII directly phosphorylate Fpr3 at CKII purified to than from S. cerevisiae was with as either or The were by and the phosphorylated proteins were and to phosphorylated Fpr3 at and tyrosine the Fpr3 phosphorylated in vitro a 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). was not phosphorylated at that Tyr184 is the site of tyrosine phosphorylation In further of the that CKII and not a was responsible for the tyrosine phosphorylation of we found that human CKII and purified also phosphorylated Fpr3 at phosphotyrosine was detected both by of and by of phosphorylated by in the of of CKII at Tyr was not E. lacks detectable protein-tyrosine kinase activity D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Thorner J. Martin G.S. J. PubMed Scopus Google Scholar), these results that CKII directly phosphorylate Fpr3 at Tyr184 and that CKII is a protein kinase with dual specificity. the of Tyr184 phosphorylation by CKII, we the of and tyrosine phosphorylation of Fpr3 in vitro. was with and and the of was at the phosphorylation at a than either Ser or phosphorylation and phosphorylation of a of of of in with findings 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). for the of Tyr be that serine or threonine phosphorylation is for efficient tyrosine serine or threonine phosphorylation Tyr184 was phosphorylated with CKII, dephosphorylated at Tyr184 by with purified we have shown previously that this all phospho-Tyr from Fpr3 its of and (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). was used as substrate for a of phosphorylation by CKII, the of tyrosine was than that with the substrate These results indicate that phosphorylation at serine or threonine residues subsequent phosphorylation at Tyr184. In Fpr3 is phosphorylated to a of of of Fpr3 (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 serine residues that are phosphorylated have not been one of is likely to be because a peptide corresponding to residues was phosphorylated at serine by not The that phospho-Ser has been reported to as a specificity determinant for CKII in vitro F. J.W. 1991; PubMed Scopus (24) Google Scholar) raised the possibility that phosphorylation of Ser186 subsequent phosphorylation of Fpr3 at Tyr184. this was the synthetic residues in which Ser186 been by or were used as in vitro substrates for The peptide was the efficient the peptide phosphorylated at a than the P-Ser The peptide no detectable Tyr These results that at position +2 Tyr phosphorylation by The results indicate that the yeast Fpr3 be phosphorylated at tyrosine by CKII from a of CKII the of a protein kinase with dual specificity. The site phosphorylated in Fpr3 is and this is phosphorylated both in vitro and in Phosphorylation at Tyr184 appears to via a in which phosphorylation at Ser186 is for subsequent phosphorylation at Tyr184. in vitro have to the that CKII phosphorylates substrates only at serine and threonine residues (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 not of phosphorylating the peptides or J.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, F. F. G. J. Biochem. PubMed Scopus Google Scholar). CKII purified from was reported to be of of tyrosine and of phosphorylation T. H. R. Biochem. 1995; Google Scholar), the of a kinase was not in and CKII purified from S. cerevisiae not display these we as reported that CKII phosphorylate the Fpr3 peptide at the site at which Fpr3 tyrosine phosphorylation in These findings that Tyr phosphorylation by CKII is and that it at to surrounding Tyr184 in is at tyrosine phosphorylation of Fpr3 by CKII is an to this or CKII also phosphorylates other proteins containing tyrosine residues within the sequence In this is no that CKII phosphorylates proteins other than Fpr3 at we at one other protein in Ptp1-deficient yeast that to be phosphorylated at tyrosine in a are a number of of or protein In many the phosphorylation of a serine by a protein kinase is a for subsequent phosphorylation by a protein the protein kinase is the phosphorylation function to the of the phosphoacceptor site in J. Biol. Chem. 1991; Full Text PDF PubMed Google example, glycogen synthase which has a substrate sequence of phosphorylates glycogen synthase at serine residues only after phosphorylation at a serine by CKII FASEB J. 1990; PubMed Scopus Google Scholar). peptide substrates to the of glycogen synthase glycogen synthase kinase-3 was shown to phosphorylate a of in an with the of A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). synthase is also phosphorylated at serine by protein kinase; this phosphorylation at position to a which be phosphorylated by protein casein kinase H. A. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar, A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The which at tyrosine residues in an provides an of phosphorylation at tyrosine residues B. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). Phosphorylation of Ser186 in Fpr3 appears to be a specificity determinant for the phosphorylation of CKII. on phosphorylation of peptides have shown that CKII as specificity vitro F. J.W. 1991; PubMed Scopus (24) Google Scholar, Biophys. Biochem. Res. 1990; PubMed Scopus Google Scholar, D.W. A. 1990; PubMed Scopus Google Scholar), to results the to CKII as a kinase in phosphorylation at Tyr in In peptides corresponding to the phosphorylated region of Fpr3, of Ser186 with either or but not with phosphorylation of that an acidic at the +2 position is even in this peptide the at and are also This specificity is in to phosphorylation by CKII, which is not dependent the of an acidic at the +2 that is an acidic at (13Tuazon T.P. Traugh J.A. Adv. Second Messenger Phosphoprotein Res. 1991; 23: 123-165PubMed Google Scholar). We a of phosphorylation at Tyr Ser for previously Fpr3, and an enhanced of tyrosine phosphorylation using as a substrate Fpr3 that was previously phosphorylated at These be by the requirement for phosphorylation at which might also to the is that might be phosphorylated as of CKII. The for the dual specificity of CKII is not The and tyrosine-specific protein kinases has in as a number of protein kinases previously have been shown to display dual specificity. example, at and is able to phosphorylate substrates at tyrosine in vitro G. R.A. Hunter T. J. Mol. Biol. Cell. 1994; 5: PubMed Scopus (53) Google Scholar). The substrate specificity of kinase is by serine phosphorylation of whereas tyrosine phosphorylation of II J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). synthase at in an in its activity J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In these and in the of CKII, tyrosine phosphorylation appears to be to a number of substrates and/or is efficient than phosphorylation by the In protein kinases with dual specificity display activity both residues the The of the catalytic domain are such that it be to an efficient at using as both and E. Hunter T. FASEB J. 1995; 9: PubMed Scopus Google Scholar). The of tyrosine phosphorylation by CKII are also Tyr184 in the of a peptide is phosphorylated to by CKII, it is only phosphorylated to in the of the vivo and in vitro 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 and this This be to by the of the tyrosine phosphorylation by CKII appears to be efficient than the of the phosphorylation of Fpr3 at Tyr184 does not the possibility that is in indicate that Fpr3 tyrosine phosphorylation as cells that tyrosine phosphorylation by CKII be a We have been to Fpr3 function is by tyrosine both because of the of physiological for Fpr3 and because only of Fpr3 is tyrosine phosphorylated even in yeast (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). we that CKII is the enzyme responsible for this it be to Fpr3 phosphorylated at Tyr184 and thus the of this phosphorylation on Fpr3 we the possibility that CKII and other protein kinases might phosphorylate tyrosine residues in an In this the of phosphotyrosine-specific such as Ptp1, be to such tyrosine of the that tyrosine phosphorylation by CKII be to this of its activity has an physiological We are to Glover for the of yeast CKII, and yeast CKII expression and to and for