FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2077808462> ?p ?o ?g. }

• W2077808462 endingPage "27815" @default.
• W2077808462 startingPage "27806" @default.
• W2077808462 abstract "Bruton's tyrosine kinase (Btk), a member of the Tec family of cytosolic kinases, is essential for B cell development and function. BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement. BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells. In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro. These residues, Tyr248, Tyr357, and Tyr462, were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo. Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I. Mutation of either Tyr248, Tyr357, or Tyr462 to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation. Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression. Bruton's tyrosine kinase (Btk), a member of the Tec family of cytosolic kinases, is essential for B cell development and function. BAP/TFII-I, a protein implicated in transcriptional regulation, is associated with Btk in B cells and is transiently phosphorylated on tyrosine following B cell receptor engagement. BAP/TFII-I is a substrate for Btk in vitro and is hyperphosphorylated on tyrosine upon coexpression with Btk in mammalian cells. In an effort to understand the physiologic consequences of BAP/TFII-I tyrosine phosphorylation following B cell receptor stimulation, site-directed mutagenesis and phosphopeptide mapping were used to locate the predominant sites of BAP/TFII-I phosphorylation by Btk in vitro. These residues, Tyr248, Tyr357, and Tyr462, were also found to be the major sites for Btk-dependent phosphorylation of BAP/TFII-I in vivo. Residues Tyr357 and Tyr462 are contained within the loop regions of adjacent helix-loop-helix-like repeats within BAP/TFII-I. Mutation of either Tyr248, Tyr357, or Tyr462 to phenylalanine reduced transcription from a c-fos promoter relative to wild-type BAP/TFII-I in transfected COS-7 cells, consistent with the interpretation that phosphorylation at these sites contributes to transcriptional activation. Phosphorylation of BAP/TFII-I by Btk may link engagement of receptors such as surface immunoglobulin to modulation of gene expression. Bruton's tyrosine kinase SH2, and SH3, Src homology 1, 2, and 3, respectively helix-loop-helix maltose-binding protein Dulbecco's modified Eagle's medium polyvinylidene difluoride thymidine kinase Bruton's tyrosine kinase (Btk),1 which is expressed in B cells and cells of the myeloid lineage, was initially identified as the target of mutations responsible for X-linked agammaglobulinemia in humans (1Tsukada S. Saffran D.C. Rawlings D.J. Parolini O. Allen R.C. Klisak I. Sparkes R.S. Kubagawa H. Mohandas T. Quan S. Belmont J. Cooper M. Conley M. Witte O. Cell. 1993; 72: 279-290Abstract Full Text PDF PubMed Scopus (1167) Google Scholar, 2Vetrie D. Vorechovsky I. Sideras P. Holland J. Davies A. Flinter F. Hammarstrom L. Kinnon C. Levinsky R. Bobrow M. Smith E. Bentley D.R. Nature. 1993; 361: 226-233Crossref PubMed Scopus (1263) Google Scholar). Patients with X-linked agammaglobulinemia exhibit an intrinsic defect in B cell development; peripheral B cells are rare and of an immature phenotype, but the number of pre-B cells in the bone marrow is not significantly reduced, consistent with impairment of the pre-B to B cell transition (3Pearl E.R. Vogler L.B. Okos A.J. Crist W.M. Lawton A.R.D. Cooper M.D. J. Immunol. 1978; 120: 1169-1175PubMed Google Scholar). A point mutation in the orthologous tyrosine kinase is responsible for an X-linked deficiency of B cell function (X-linked immunodeficiency, or xid) in the mouse (4Rawlings D.J. Saffran D.C. Tsukada S. Largaespada D.A. Grimaldi J.C. Cohen L. Mohr R.N. Bazan J.F. Howard M. Copeland N.G. Jenkins N.A. Witte O. Science. 1993; 261: 358-361Crossref PubMed Scopus (781) Google Scholar, 5Thomas J.D. Sideras P. Smith C.I. Vorechovsky I. Chapman V. Paul W.E. Science. 1993; 261: 355-358Crossref PubMed Scopus (578) Google Scholar). The mouse xid phenotype is distinct from X-linked agammaglobulinemia (6Scher I. Adv. Immunol. 1982; 33: 1-71Crossref PubMed Scopus (266) Google Scholar). Antibody responses to some T cell-independent antigens are absent, but responses to most T cell-dependent antigens are intact. Peripheral B cells are slightly reduced and skewed toward an immature phenotype. Survival of peripheral B cells is diminished, and the peritoneal B-1 B cell population is absent. Notably, xid mice do not exhibit the block in early B cell development observed in patients with X-linked agammaglobulinemia. Btk null mice exhibit a phenotype resembling xid, suggesting that B cell development has a more stringent requirement for Btk in humans than in mice (7Hardy R.R. Hayakawa K. Parks D.R. Herzenberg L.A. Nature. 1983; 306: 270-272Crossref PubMed Scopus (116) Google Scholar, 8Hayakawa K. Hardy R.R. Herzenberg L.A. Eur. J. Immunol. 1986; 16: 450-456Crossref PubMed Scopus (289) Google Scholar, 9Perlmutter R.M. Nahm M. Stein K.E. Slack J. Zitron I. Paul W.E. Davie J.M. J. Exp. Med. 1979; 149: 993-998Crossref PubMed Scopus (87) Google Scholar, 10Khan W.N. Nilsson A. Mizoguchi E. Castigli E. Forsell J. Bhan A.K. Geha R. Sideras P. Alt F.W. Int. Immunol. 1997; 9: 395-405Crossref PubMed Scopus (55) Google Scholar). Btk belongs to the Tec family of cytosolic protein-tyrosine kinases, which includes Btk, Itk, Tec, and Bmx. This group of kinases is related to the Src family by the presence of SH3, SH2, and SH1 (catalytic) domains. It is distinguished from the Src family by 1) the presence of pleckstrin homology and Tec homology domains, which serve as binding sites for phospholipids and SH3 domains, respectively; 2) the absence of an N-terminal myristoylation site; and 3) the lack of a regulatory tyrosine residue near the carboxyl terminus (11Siliciano J.D. Morrow T.A. Desiderio S.V. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11194-11198Crossref PubMed Scopus (236) Google Scholar, 12Mano H. Mano K. Tang B. Koehler M. Yi T. Gilbert D.J. Jenkins N.A. Copeland N.G. Ihle J.N. Oncogene. 1993; 8: 417-424PubMed Google Scholar, 13Tamagnone L. Lahtinen I. Mustonen T. Virtaneva K. Francis F. Muscatelli F. Alitalo R. Smith C.I. Larsson C. Alitalo K. Oncogene. 1994; 9: 3683-3688PubMed Google Scholar, 14Vihinen M. Nilsson L. Smith C.I. FEBS Lett. 1994; 350: 263-265Crossref PubMed Scopus (90) Google Scholar). An atypical member of the Tec family, Rlk/Txk, lacks a pleckstrin homology domain but has 54–62% amino acid identity to Btk in the remainder of its sequence (15Haire R.N. Ohta Y. Lewis J.E. Fu S.M. Kroisel P. Litman G.W. Hum. Mol. Genet. 1994; 3: 897-901Crossref PubMed Scopus (83) Google Scholar, 16Hu Q. Davidson D. Schwartzberg P.L. Macchiarini F. Lenardo M.J. Bluestone J.A. Matis L.A. J. Biol. Chem. 1995; 270: 1928-1934Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Btk has been implicated as important in signaling from the B cell receptor for antigen, the interleukin-5 and -6 receptors, and CD38 on B cells (17Koike M. Kikuchi Y. Tominaga A. Takaki S. Akagi K. Miyazaki J. Yamamura K. Takatsu K. Int. Immunol. 1995; 7: 21-30Crossref PubMed Scopus (46) Google Scholar, 18Matsuda T. Takahashi-Tezuka M. Fukada T. Okuyama Y. Fujitani Y. Tsukada S. Mano H. Hirai H. Witte O.N. Hirano T. Blood. 1995; 85: 627-633Crossref PubMed Google Scholar, 19Santos-Argumedo L. Lund F.E. Heath A.W. Solvason N. Wu W.W. Grimaldi J.C. Parkhouse R.M. Howard M. Int. Immunol. 1995; 7: 163-170Crossref PubMed Scopus (90) Google Scholar); the FcεRI receptor on mast cells (20Kawakami Y. Yao L. Miura T. Tsukada S. Witte O.N. Kawakami T. Mol. Cell. Biol. 1994; 14: 5108-5113Crossref PubMed Google Scholar); the FcγRI on macrophages; and the thrombin receptor on platelets (21Rawlings D.J. Clin. Immunol. 1999; 91: 243-253Crossref PubMed Scopus (87) Google Scholar). Upon engagement of the B cell receptor, Btk is phosphorylated by the tyrosine kinase Lyn at residue Tyr551. This permits Btk to undergo autophosphorylation at residue Tyr223, after which kinase activity is increased (22Rawlings D.J. Scharenberg A.M. Park H. Wahl M.I. Lin S. Kato R.M. Fluckiger A.C. Witte O.N. Kinet J.P. Science. 1996; 271: 822-825Crossref PubMed Scopus (381) Google Scholar). The activity of Btk can also be modulated by association with Gqα proteins (23Bence K. Ma W. Kozasa T. Huang X.Y. Nature. 1997; 389: 296-299Crossref PubMed Scopus (169) Google Scholar), Gβγ proteins (24Langhans-Rajasekaran S.A. Wan Y. Huang X.Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8601-8605Crossref PubMed Scopus (127) Google Scholar), or phosphatidylinositol phosphates (25Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (388) Google Scholar). A fraction of Btk coimmunoprecipitates with a protein of ∼135 kDa termed BAP (for Btk-associated protein); based on this association, BAP was purified, and its cDNA was molecularly cloned (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar). BAP is identical to the putative transcription factor TFII-I (27Roy A.L. Du H. Gregor P.D. Novina C.D. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Crossref PubMed Scopus (171) Google Scholar), which was identified by its ability to stimulate transcription from initiator elements (27Roy A.L. Du H. Gregor P.D. Novina C.D. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Crossref PubMed Scopus (171) Google Scholar) and its synergy with Phox I and serum response factor in enhancing transcription from the c-fos promoter (28Grueneberg D.A. Henry R.W. Brauer A. Novina C.D. Cheriyath V. Roy A.L. Gilman M. Genes Dev. 1997; 11: 2482-2493Crossref PubMed Scopus (119) Google Scholar). A distinctive feature of BAP/TFII-I is the occurrence of six helix-loop-helix (HLH)-like repeats. In contrast to typical HLH motifs, however, which contain loop regions of between 6 and 20 amino acids (29Ferre-D'Amare A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar), the HLH-like repeats of BAP/TFII-I contain extended loop regions of ∼70 amino acids (27Roy A.L. Du H. Gregor P.D. Novina C.D. Martinez E. Roeder R.G. EMBO J. 1997; 16: 7091-7104Crossref PubMed Scopus (171) Google Scholar). Four isoforms of BAP/TFII-I, generated by alternative splicing of primary RNA transcripts, are differentially expressed in various tissues. Amino acid sequence differences among these four isoforms are confined to the interval between the first and second HLH-like domains (30Cheriyath V. Roy A.L. J. Biol. Chem. 2000; 275: 26300-26308Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). 2H. Taylor, and S. Desiderio, unpublished results. Two sequence motifs resembling the Src kinase autophosphorylation consensus sequence (EDXDY) are present within this interval and are preserved in all four BAP/TFII-I isoforms. BAP/TFII-I is transiently phosphorylated on tyrosine following B cell receptor engagement, with kinetics that closely follow tyrosine phosphorylation of Btk (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar). Increased tyrosine phosphorylation of BAP/TFII-I is also observed upon cotransfection with Btk into fibroblastoid cells. This response is dependent upon Btk kinase activity, since cotransfection with the kinase-inactive mutant Btk(K430E) fails to enhance tyrosine phosphorylation of BAP/TFII-I (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar). These observations and the physical association of Btk with BAP/TFII-I in B lymphoid cells (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar) suggested that BAP/TFII-I is a physiologic substrate for phosphorylation by Btk following B cell receptor stimulation. Several lines of evidence suggest that phosphorylation modulates the ability of BAP/TFII-I to stimulate transcription. First, dephosphorylation impairs the ability of BAP/TFII-I to stimulate transcription from a Vβ promoter in vitro, while sparing its ability to bind the Vβ initiator element (31Novina C.D. Cheriyath V. Roy A.L. J. Biol. Chem. 1998; 273: 33443-33448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Second, stimulation of Vβ transcription by BAP/TFII-I in transfected cells is enhanced by wild-type Btk but not by a kinase-inactive mutant (32Novina C.D. Kumar S. Bajpai U. Cheriyath V. Zhang K. Pillai S. Wortis H.H. Roy A.L. Mol. Cell. Biol. 1999; 19: 5014-5024Crossref PubMed Scopus (94) Google Scholar). Third, the transcriptional activity of BAP/TFII-I in transfected cells is impaired by mutation of a site conforming to the Src autophosphorylation consensus (31Novina C.D. Cheriyath V. Roy A.L. J. Biol. Chem. 1998; 273: 33443-33448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Fourth, epidermal growth factor, which enhances c-fos promoter activity, induces tyrosine phosphorylation of BAP/TFII-I (33Kim D.W. Cheriyath V. Roy A.L. Cochran B.H. Mol. Cell. Biol. 1998; 18: 3310-3320Crossref PubMed Scopus (94) Google Scholar). While these observations are consistent with the interpretation that Btk activates BAP/TFII-I by tyrosine phosphorylation, definitive evidence is lacking. As an essential step in defining a regulatory relationship between Btk and BAP/TFII-I, we have mapped the major Btk phosphorylation sites in BAP/TFII-I. Bacterially expressed, purified Btk phosphorylates BAP/TFII-I at three predominant sites: Tyr248, Tyr357, and Tyr462. The first site lies in the interrepeat region between the first and second HLH-like domains; the two other sites are at analogous positions in putative loop regions of the second and third HLH-like domains. When Btk is coexpressed with BAP/TFII-I in mammalian cells, BAP/TFII-I is specifically hyperphosphorylated at the same three sites. Btk-specific hyperphosphorylation of a BAP/TFII-I fragment in vivo was eliminated by mutation of these sites to phenylalanine. The ability of BAP/TFII-I to stimulate expression of a c-fos reporter, in COS-7 cells was impaired by mutation of Tyr248, Tyr357, or Tyr462 to phenylalanine, consistent with the interpretation that phosphorylation of these sites plays a role in transcriptional activation. Phosphorylation of BAP/TFII-I by Btk, and perhaps other Tec-related kinases, may link engagement of receptors such as surface immunoglobulin to modulation of gene expression. Plasmid pBAPmyc, encoding the shortest isoform of BAP/TFII-I (BAP/TFII-I A), fused at the carboxyl terminus to a Pro-Gly linker and the c-Myc epitope EQKLISEEDL, was constructed from a BAP cDNA clone (26Yang W. Desiderio S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 604-609Crossref PubMed Scopus (159) Google Scholar) by polymerase chain reaction. The plasmid pBAPmyc(BN) was generated from pBAPmyc by introduction of SalI, XbaI, and in-frameBamHI sites 5′ of the BAP/TFII-I coding sequence. The following BAP/TFII-I carboxyl- and amino-truncations were constructed from pBAPmyc(BN) using polymerase chain reaction: pBAP-N-1R (residues 1–189), pBAP-N-NR2 (residues 1–303), pBAP-N-2R (residues 1–398), pBAP-N-3R (residues 1–503), pBAP-4R-CT (residues 514–957), and pBAP-5R-CT (residues 675–957). All constructs share an identical initiation sequence (34Kozak M. Mol. Cell. Biol. 1989; 9: 5073-5080Crossref PubMed Scopus (374) Google Scholar) and a carboxyl-terminal c-Myc epitope. Tyrosine to phenylalanine point mutations were introduced using divergent polymerase chain reaction (35Jones D.H. Winistorfer S.C. BioTechniques. 1992; 12 (, 532, 534–535): 528-530PubMed Google Scholar). The identities of mutant constructs were verified by nucleotide sequence analysis. For expression of c-Myc-tagged BAP/TFII-I and BAP truncation proteins in Escherichia coli under inducible control of thearaBAD promoter, the BamHI/EcoRV fragments of pBAPmyc(BN) and its derivatives, encoding full-length BAP/TFII-I or BAP/TFII-I truncation proteins, were inserted between theBglII and PvuII sites of pBADHisB (Invitrogen). For eukaryotic expression studies, the SalI/NotI fragments of pBAPmyc(BN) and its derivatives, encoding full-length or truncated BAP/TFII-I proteins, were inserted between theXhoI and NotI sites of pCIS2 (36Gorman C.M. Gies D.R. McCray G. DNA Protein Eng. Tech. 1990; 2: 3-10Google Scholar). Btk pCIS2 and Btk mutant K430E pCIS2 have been described (37Yang W. Malek S.N. Desiderio S. J. Biol. Chem. 1995; 270: 20832-20840Crossref PubMed Scopus (51) Google Scholar). Plasmids for bacterial expression of Btk (pMal-C2Btk) or Btk(K430E) (pMal-C2Bke), fused at the amino terminus to maltose-binding protein (MBP), were constructed by inserting the 2-kilobase pairBamHI fragments of pCIS2Btk and pCIS2Btk(K430E) into theBamHI site of pMal-C2 (New England Biolabs). A reporter construct containing the murine c-fos wild-type promoter (33Kim D.W. Cheriyath V. Roy A.L. Cochran B.H. Mol. Cell. Biol. 1998; 18: 3310-3320Crossref PubMed Scopus (94) Google Scholar) was a kind gift of D. Kim and B. Cochran. TheHindII fragment containing the c-fos promoter was cloned into the HindII site upstream of the firefly luciferase gene within the Promega PGL3 basic vector, and orientation was confirmed by sequence analysis. The herpes simplex thymidine kinaseRenilla vector (pRL-TK) was purchased from Promega. The human embryonic kidney fibroblast cell line 293 and the simian fibroblast cell line COS-7 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 50 µg/ml streptomycin, and 50 units/ml penicillin. E. coli BL21, transformed with either pMal-C2Btk or pMal-C2Bke, was cultured overnight at 37° C in 50 ml of rich medium containing 2 g/liter glucose and 50 µg/ml carbenicillin. The overnight cultures were diluted 1:20 in the same medium and grown at 37 °C until A 600was 0.5. Expression was then induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to 0.3 mm, and incubation was continued for 3 h at 37 °C. Cells were collected by centrifugation and resuspended in 25 ml of buffer F (50 mm HEPES (pH 7.5), 10 mmMgCl2, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 2 µg/ml chymostatin, 2 µg/ml pepstatin, and 2 µg/ml antipain) per 500-ml culture. Lysozyme and phenylmethylsulfonyl fluoride were added to 1 mg/ml and 1 mm, respectively. After incubation for 30 min on ice, bacterial suspensions were frozen in liquid nitrogen and stored at −80 °C. MBP-Btk and MBP-Btk(K430E) fusion proteins were purified by amylose affinity chromatography (New England Biolabs). Frozen bacterial suspensions were thawed in a 30 °C water bath with gentle agitation and then placed on ice. Ice-cold EDTA was added for a final concentration of 2 mm, and EGTA was added for a final concentration of 10 mm. After mixing by inversion, the lysate was sonicated for three cycles of 10 pulses each with a Branson microtip sonifier (power output 5, duty cycle 50%). An equal volume (25 ml) of lysate dilution buffer (300 mm NaCl, 100 mm Tris-HCl (pH 7.4), 10 mm EGTA, 2 mm EDTA, 0.2 mm dithiothreitol, 2% Nonidet P-40, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 2 µg/ml chymostatin, 2 µg/ml pepstatin, and 2 µg/ml antipain) was added to 25 ml of bacterial suspension. Suspensions were mixed by inversion, incubated on ice for 20 min, and then clarified by centrifugation at 30,000 × g for 20 min at 4 °C. The supernatant was loaded twice over a 2-ml amylose resin, pre-equilibrated with wash buffer 1 (150 mm NaCl, 50 mm Tris-HCl (pH 7.4), 10 mm EGTA, 2 mm EDTA, 0.1 mmdithiothreitol, 1% Nonidet P-40, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 2 µg/ml chymostatin, 2 µg/ml pepstatin, and 2 µg/ml antipain). The column was washed sequentially with 10 ml of wash buffer 1, 10 ml of wash buffer 1 supplemented to 1 m NaCl, and 10 ml of wash buffer 2 (100 mm NaCl, 50 mmTris-HCl (pH 7.4), 0.1 mm dithiothreitol, 10% glycerol). Bound protein was eluted with 3 ml of elution buffer (100 mm NaCl, 50 mm Tris-HCl (pH 7.4), 10 mm maltose, 0.1 mm dithiothreitol, and 10% glycerol). Eluates were dialyzed against elution buffer lacking maltose, frozen in liquid nitrogen, and stored at −80 °C. Wild-type and mutant BAP/TFII-I fragments, tagged at the amino terminus with polyhistidine, were expressed from the BAP pBAD HIS constructs in E. coli (TOP10; Invitrogen). Bacterial cultures were grown overnight at 37 °C in 2× YT containing 50 µg/ml carbenicillin, diluted 1:10 in the same medium, and incubated for 1 h at 37 °C. Expression was induced by the addition of arabinose to 0.002% (w/v) and further incubation for 4 h at 37 °C. Cells were collected by centrifugation and resuspended in buffer F (25 ml per 500-ml culture). After the addition of lysozyme to 1 mg/ml and phenylmethylsulfonyl fluoride to 1 mm, bacterial suspensions were incubated on ice for 30 min, frozen in liquid nitrogen, and stored at −80 °C. Bacterial suspensions were thawed with gentle agitation in a 30 °C water bath and placed on ice immediately upon thawing. Samples were sonicated for three cycles of 10 pulses each with a Branson microtip sonifier (power output 5, duty cycle 50%). One volume of 2× imidazole wash buffer (500 mm NaCl, 100 mm Tris-HCl (pH 7.4) and 40 mm imidazole) containing 0.4% Tween 20 was added, and samples were incubated on ice for 20 min. Lysis was completed by sonication for two cycles of 10 pulses each (output 5, duty cycle 50%). Lysates were clarified by centrifugation at 30,000 × g for 30 min at 4 °C, diluted 2-fold with imidazole wash buffer (250 mm NaCl, 50 mmTris-HCl (pH 7.4), 20 mm imidazole) and loaded twice onto Ni2+-nitrilotriacetic acid (Invitrogen). Columns were washed with 50 bed volumes of imidazole wash buffer, followed by 2 volumes of imidazole pre-elution buffer (250 mm NaCl, 50 mm Tris-HCl (pH 7.4), 25 mm imidazole, 15% glycerol). Samples were eluted with 1 bed volume of imidazole elution buffer (300 mm NaCl, 250 mm imidazole, 50 mm Tris-HCl (pH 7.4), and 10% glycerol); dialyzed against a buffer containing 50 mm NaCl, 50 mm Tris-HCl (pH 7.4), 1 mmphenylmethylsulfonyl fluoride, 0.1 mm dithiothreitol, and 10% glycerol; frozen in liquid nitrogen; and stored at −80 °C. Purified BAP/TFII-I substrate was combined with 20 µl of 2× kinase buffer (40 mm Tris-HCl (pH 7.2), 4 mm MnCl2, 2 mmNa2MoO4), 6 µl of distilled H2O, and 10 µCi of [γ-32P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences). Purified MBP-Btk or MBP-Btk(K430E) (2 µg in 5 µl) was added to a total volume of 40 µl, and samples were incubated at 37 °C for 30 min. Reactions were terminated by the addition of SDS-polyacrylamide gel loading buffer. Products were fractionated by gel electrophoresis and transferred to PVDF (Millipore Corp.) for tryptic phosphopeptide mapping. Transient transfections were performed by the calcium phosphate method (38Paborsky L.R. Fendly B.M. Fisher K.L. Lawn R.M. Marks B.J. McCray G. Tate K.M. Vehar G.A. Gorman C.M. Protein Eng. 1990; 3: 547-553Crossref PubMed Scopus (35) Google Scholar). 293 cells were transiently transfected with 5 µg of wild-type or mutant BAP/TFII-I expression constructs in pCIS2, 5 µg of pCIS2Btk or pCIS2Btk(K430E), and 1 µg of pRSVT. At 40 h after transfection, cells were labeled for 4 h at 37 °C with 250 µCi/ml [32P]orthophosphate in phosphate-free DMEM supplemented with 10% dialyzed fetal bovine serum, 50 µg/ml streptomycin, and 50 units/ml penicillin. Cells were treated for 10 min at 37 °C with 1 mm sodium pervanadate, washed once with 5 ml of cold PBS containing 1 mm Na3VO4 and 1 mm EDTA, collected by centrifugation, frozen on dry ice, and stored at −80 °C. Cells were lysed in radioimmune precipitation buffer (150 mm NaCl, 50 mm Tris-HCl (pH 8.0), 1 mmNa3VO4, 1 mmNa2MoO4, 1 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2.5 µg/ml pepstatin, 2.5 µg/ml antipain, and 1 µg/ml chymostatin) for 30 min on ice. Lysates were clarified by centrifugation at 15,000 × g for 30 min at 4 °C. Epitope-tagged BAP/TFII-I was immunoprecipitated by incubation with anti-c-Myc monoclonal antibody 9E10 for 1 h on ice. Following the addition of 25 µl of protein A/G-Sepharose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), suspensions were rocked for 1 h at 4 °C. Beads were washed three times for 10 min each in radioimmune precipitation buffer; immunoprecipitated proteins were solubilized in SDS-loading buffer and resolved by polyacrylamide gel electrophoresis. Protein was transferred to PVDF (Millipore) for tryptic phosphopeptide mapping. Tryptic phosphopeptides were prepared according to methods previously described (39Hunter T. Sefton B.M. Methods Enzymol. 1991; 210: 110-153Google Scholar) and analyzed by alkaline gel electrophoresis (40West M.H.P. Wu R.S. Bonner W.M. Electrophoresis. 1984; 5: 133-138Crossref Scopus (74) Google Scholar). Briefly, PVDF membrane slices containing phosphoproteins were excised, washed with water, and then soaked in 0.5% polyvinylpyrolidone (Sigma) for 30 min at 37 °C. Membranes were then washed five times with water and once with freshly prepared 0.05 mNH4HCO3. Immobilized proteins were digested with 1 ml of 0.3 mg/ml trypsin (l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, Sigma) in 0.05 mNH4HCO3 overnight at 37 °C. Supernatants were dried under vacuum and washed three times in water. Dried pellets were dissolved in 10 µl of sample buffer (0.125 mTris-HCl (pH 6.8), 6 m urea) and fractionated on a 40% alkaline gel as described (40West M.H.P. Wu R.S. Bonner W.M. Electrophoresis. 1984; 5: 133-138Crossref Scopus (74) Google Scholar). Phosphopeptides were detected by a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). The ability of BAP/TFII-I to stimulate transcription from the c-fospromoter was assessed as previously described (33Kim D.W. Cheriyath V. Roy A.L. Cochran B.H. Mol. Cell. Biol. 1998; 18: 3310-3320Crossref PubMed Scopus (94) Google Scholar) with modifications. COS-7 cells, grown to 80–90% confluence, were trypsinized, resuspended in DMEM containing 10% FBS and 0.1 mmnonessential amino acids (NEAA), and seeded in 24-well plates at 6 × 104 cells/well. Cells were transfected 16–20 h later using LipofectAMINE 2000 (Life Technologies, Inc.). Each well was transfected with 0.1 µg of c-fos in PGL3, 0.1 µg of pRL-TK, and 0.6 µg of either pCIS2 or wild-type or mutant BAPmyc in pCIS2. For each well, 1 µl of LipofectAMINE 2000 was diluted in 50 µl of OPTI-MEM (Life Technologies), incubated for 5 min at room temperature, and then combined with DNA diluted in 50 µl of OPTI-MEM. Complexes were allowed to form for 30 min at room temperature and then added to each well, which contained 250 ml of fresh DMEM containing 0.1 mm NEAA. After 5 h at 37 °C, medium was replaced with DMEM supplemented with 0.5% FBS, 50 µg/ml streptomycin, and 50 units/ml penicillin. At 36–40 h after transfection, cells were stimulated with 10% FBS for 5 h. Cells were then lysed in 100 ml of passive lysis buffer and assayed for firefly and Renilla luciferase activity using the Promegadual luciferase kit, and c-fos reporter activity was assessed as the ratio of firefly to Renilla activities. The effects of Btk and BAP/TFII-I on the activity of the c-fospromoter were assessed as above, except that transfections contained 0.4 µg of pCIS2 vector or pCIS2-BAP/TFII-I and 0.3 µg of pCIS2Btk or pCIS2Btk(K430E). The overall strategy used in these phosphorylation site mapping studies was 1) to determine which portions of BAP/TFII-I are phosphorylated by Btk in vitro; 2) to define the specific tyrosine residues phosphorylated by Btk in vitro by a combination of phosphopeptide mapping and site-directed mutagenesis; and 3) to verify that the same tyrosine residues are hyperphosphorylated in vivo upon coexpression of BAP/TFII-I with active Btk. The shortest BAP/TFII-I isoform, BAP/TFII-I A, contains all 24 tyrosine residues present in the larger isoforms (Fig.1A).2 This isoform and its derivative polypeptide fragments were therefore used forin vitro phosphorylation site mapping. The following BAP/TFII-I protein fragments, fused at the amino terminus to polyhistidine and the carboxyl terminus to a c-Myc epitope, were expressed in bacterial cells and purified by Ni2+-nitrilotriacetic acid affinity chromatography (Fig.1 B): BAP N-3R (residues 1–503), BAP N-2R (residues 1–398), BAP N-NR2 (residues 1–303), BAP N-1R (residues 1–189), BAP 4R-CT (residues 514–957), and BAP 5R-CT (residues 675–957). Wild-type BAP/TFII-I, tagged with polyhistidine and a c-Myc epitope, was expressed and purified in parallel. To define the sites at which Btk phosphorylates BAP/TFII-I in vitro, it was essential to obtain active Btk in a form free of contaminating tyrosine kinases. Btk immunoprecipitated from mammalian cells was unsatisfactory for this purpose, since pilot experiments with a kinase-inactive mutant Btk revealed the presence of additional tyrosine kinase activities in such preparations. Recovery of catalytically active Btk, expressed inE. coli, has been demonstrated (23Bence K. Ma W. Kozasa T. Huang X.Y. Nature. 1997; 389: 296-299Crossref PubMed Scopus (169) Google Scholar). We therefore employed recomb" @default.
• W2077808462 created "2016-06-24" @default.
• W2077808462 creator A5022139249 @default.
• W2077808462 creator A5026873741 @default.
• W2077808462 date "2001-07-01" @default.
• W2077808462 modified "2023-09-28" @default.
• W2077808462 title "Identification of Phosphorylation Sites for Bruton's Tyrosine Kinase within the Transcriptional Regulator BAP/TFII-I" @default.
• W2077808462 cites W1529773650 @default.
• W2077808462 cites W1592751942 @default.
• W2077808462 cites W1964426040 @default.
• W2077808462 cites W1966408108 @default.
• W2077808462 cites W1967703786 @default.
• W2077808462 cites W1974521227 @default.
• W2077808462 cites W1974617606 @default.
• W2077808462 cites W1982350284 @default.
• W2077808462 cites W1984671408 @default.
• W2077808462 cites W1993807543 @default.
• W2077808462 cites W1994816231 @default.
• W2077808462 cites W2003955301 @default.
• W2077808462 cites W2007750331 @default.
• W2077808462 cites W2015597774 @default.
• W2077808462 cites W2020268217 @default.
• W2077808462 cites W2031616290 @default.
• W2077808462 cites W2035277099 @default.
• W2077808462 cites W2041121472 @default.
• W2077808462 cites W2054526083 @default.
• W2077808462 cites W2056030264 @default.
• W2077808462 cites W2065959166 @default.
• W2077808462 cites W2069625012 @default.
• W2077808462 cites W2080251738 @default.
• W2077808462 cites W2081296266 @default.
• W2077808462 cites W2090494599 @default.
• W2077808462 cites W2100034730 @default.
• W2077808462 cites W2101244702 @default.
• W2077808462 cites W2112568143 @default.
• W2077808462 cites W2113764179 @default.
• W2077808462 cites W2116705053 @default.
• W2077808462 cites W2138303004 @default.
• W2077808462 cites W2144938320 @default.
• W2077808462 cites W2145592992 @default.
• W2077808462 cites W2165742826 @default.
• W2077808462 cites W2416378933 @default.
• W2077808462 cites W2425942719 @default.
• W2077808462 doi "https://doi.org/10.1074/jbc.m103692200" @default.
• W2077808462 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11373296" @default.
• W2077808462 hasPublicationYear "2001" @default.
• W2077808462 type Work @default.
• W2077808462 sameAs 2077808462 @default.
• W2077808462 citedByCount "26" @default.
• W2077808462 countsByYear W20778084622012 @default.
• W2077808462 countsByYear W20778084622015 @default.
• W2077808462 countsByYear W20778084622017 @default.
• W2077808462 countsByYear W20778084622019 @default.
• W2077808462 countsByYear W20778084622020 @default.
• W2077808462 countsByYear W20778084622021 @default.
• W2077808462 crossrefType "journal-article" @default.
• W2077808462 hasAuthorship W2077808462A5022139249 @default.
• W2077808462 hasAuthorship W2077808462A5026873741 @default.
• W2077808462 hasBestOaLocation W20778084621 @default.
• W2077808462 hasConcept C104317684 @default.
• W2077808462 hasConcept C116834253 @default.
• W2077808462 hasConcept C11960822 @default.
• W2077808462 hasConcept C153911025 @default.
• W2077808462 hasConcept C185592680 @default.
• W2077808462 hasConcept C27153228 @default.
• W2077808462 hasConcept C2776165026 @default.
• W2077808462 hasConcept C42362537 @default.
• W2077808462 hasConcept C55493867 @default.
• W2077808462 hasConcept C59822182 @default.
• W2077808462 hasConcept C62478195 @default.
• W2077808462 hasConcept C6929976 @default.
• W2077808462 hasConcept C86339819 @default.
• W2077808462 hasConcept C86803240 @default.
• W2077808462 hasConcept C90059517 @default.
• W2077808462 hasConcept C95444343 @default.
• W2077808462 hasConceptScore W2077808462C104317684 @default.
• W2077808462 hasConceptScore W2077808462C116834253 @default.
• W2077808462 hasConceptScore W2077808462C11960822 @default.
• W2077808462 hasConceptScore W2077808462C153911025 @default.
• W2077808462 hasConceptScore W2077808462C185592680 @default.
• W2077808462 hasConceptScore W2077808462C27153228 @default.
• W2077808462 hasConceptScore W2077808462C2776165026 @default.
• W2077808462 hasConceptScore W2077808462C42362537 @default.
• W2077808462 hasConceptScore W2077808462C55493867 @default.
• W2077808462 hasConceptScore W2077808462C59822182 @default.
• W2077808462 hasConceptScore W2077808462C62478195 @default.
• W2077808462 hasConceptScore W2077808462C6929976 @default.
• W2077808462 hasConceptScore W2077808462C86339819 @default.
• W2077808462 hasConceptScore W2077808462C86803240 @default.
• W2077808462 hasConceptScore W2077808462C90059517 @default.
• W2077808462 hasConceptScore W2077808462C95444343 @default.
• W2077808462 hasIssue "30" @default.
• W2077808462 hasLocation W20778084621 @default.
• W2077808462 hasOpenAccess W2077808462 @default.
• W2077808462 hasPrimaryLocation W20778084621 @default.
• W2077808462 hasRelatedWork W138827836 @default.
• W2077808462 hasRelatedWork W1988628626 @default.
• W2077808462 hasRelatedWork W2019151336 @default.

• next