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• W2039659863 abstract "Bruton's tyrosine kinase (BTK) is a member of the Tec family of kinases, which is a subgroup of the nonreceptor cytoplasmic protein tyrosine kinases. BTK has been shown to be important in the proliferation, differentiation, and signal transduction of B cells. Mutations in BTK result in B cell immune deficiency disorders, such as X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Although BTK plays multiple roles in the life of a B cell, its functional role in neuronal cells has not been elucidated. In the present study, we demonstrate that BTK activates transcription factor, cAMP response element (CRE)-binding protein (CREB), and subsequent CRE-mediated gene transcription during basic fibroblast growth factor (bFGF)-induced neuronal differentiation in immortalized hippocampal progenitor cells (H19-7). The kinase activity of BTK is also induced by bFGF, and BTK directly phosphorylates CREB at Ser-133 residue, indicating that BTK has a dual protein kinase activity. In addition, blockading BTK activation significantly inhibits CREB phosphorylation as well as the neurite outgrowth induced by bFGF in H19-7 cells. These results suggest that the activation of BTK and the subsequent phosphorylation of CREB at Ser-133 are important in the neuronal differentiation of hippocampal progenitor cells. Bruton's tyrosine kinase (BTK) is a member of the Tec family of kinases, which is a subgroup of the nonreceptor cytoplasmic protein tyrosine kinases. BTK has been shown to be important in the proliferation, differentiation, and signal transduction of B cells. Mutations in BTK result in B cell immune deficiency disorders, such as X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. Although BTK plays multiple roles in the life of a B cell, its functional role in neuronal cells has not been elucidated. In the present study, we demonstrate that BTK activates transcription factor, cAMP response element (CRE)-binding protein (CREB), and subsequent CRE-mediated gene transcription during basic fibroblast growth factor (bFGF)-induced neuronal differentiation in immortalized hippocampal progenitor cells (H19-7). The kinase activity of BTK is also induced by bFGF, and BTK directly phosphorylates CREB at Ser-133 residue, indicating that BTK has a dual protein kinase activity. In addition, blockading BTK activation significantly inhibits CREB phosphorylation as well as the neurite outgrowth induced by bFGF in H19-7 cells. These results suggest that the activation of BTK and the subsequent phosphorylation of CREB at Ser-133 are important in the neuronal differentiation of hippocampal progenitor cells. Growth factors act by binding to cell surface receptors to elicit the regulation of cell growth and differentiation (1.Huang E.J. Reichardt L.F. Annu. Rev. Neurosci. 2001; 24: 677-736Crossref PubMed Scopus (3207) Google Scholar). This, in turn, triggers a variety of intracellular signaling pathways that ultimately control cell physiology. The activation of signaling cascades changes gene expression patterns through the functional modulation of various transcription factors. These processes allow cells to coordinate long term physiological adaptation. cAMP response element-binding protein (CREB) 1The abbreviations used are: CREB, cAMP-responsive element-binding protein; bFGF, basic fibroblast growth factor; BTK, Bruton's tyrosine kinase; ca, constitutive active; CRE, cAMP-responsive element; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; GST, glutathione S-transferase; Luc, luciferase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PH domain, pleckstrin homology domain; PI-3K, phosphatidylinositol 3-kinase; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyridine; SH domain, Src homology domain; siRNA, short RNA interference; TK, thymidine kinase.1The abbreviations used are: CREB, cAMP-responsive element-binding protein; bFGF, basic fibroblast growth factor; BTK, Bruton's tyrosine kinase; ca, constitutive active; CRE, cAMP-responsive element; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GFP, green fluorescent protein; GST, glutathione S-transferase; Luc, luciferase; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PH domain, pleckstrin homology domain; PI-3K, phosphatidylinositol 3-kinase; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyridine; SH domain, Src homology domain; siRNA, short RNA interference; TK, thymidine kinase. is a central transcription factor that mediates cAMP- and calcium-dependent gene expression through the cAMP response element (2.Shaywitz A.J. Greenberg M.E. Annu. Rev. Biochem. 1999; 68: 821-861Crossref PubMed Scopus (1748) Google Scholar). Moreover, CREB activity is regulated by multiple kinases after various kinds of stimulation. Many Ser/Thr kinases can phosphorylate CREB, including protein kinase C (3.Xie H. Rothstein T.L. J. Immunol. 1995; 154: 1717-1723PubMed Google Scholar), Ca2+/calmodulin-dependent protein kinases (4.Matthews R.P. Guthrie C.R. Wailes L.M. Zhao X. Means A.R. McKnight G.S. Mol. Cell. Biol. 1994; 14: 6107-6116Crossref PubMed Scopus (492) Google Scholar, 5.Sheng M. Thompson M.A. Greenberg M.E. Science. 1991; 252: 1427-1430Crossref PubMed Scopus (1267) Google Scholar), Ras-dependent p105 kinase (6.Ginty D.D. Bonni A. Greenberg M.E. Cell. 1994; 77: 713-725Abstract Full Text PDF PubMed Scopus (673) Google Scholar), p90rsk (7.Bohm M. Moellmann G. Cheng E. Alvarez-Franco M. Wagner S. Sassone-Corsi P. Halaban R. Cell Growth Differ. 1995; 6: 291-302PubMed Google Scholar), and Rsk2 (8.Xing J. Ginty D.D. Greenberg M.E. Science. 1996; 273: 959-963Crossref PubMed Scopus (1079) Google Scholar). CREB is believed to be necessary for long term potentiation in invertebrates and vertebrates (9.Bailey C.H. Bartsch D. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13445-13452Crossref PubMed Scopus (607) Google Scholar). Furthermore, the activation of CREB plays an important role in neuronal differentiation (10.Ghil S.H. Kim B.J. Lee Y.D. Suh-Kim H. J. Neurochem. 2000; 74: 151-158Crossref PubMed Scopus (56) Google Scholar, 11.Shimomura A. Okamoto Y. Hirata Y. Kobayashi M. Kawakami K. Kiuchi K. Wakabayashi T. Hagiwara M. J. Neurochem. 1998; 70: 1029-1034Crossref PubMed Scopus (34) Google Scholar). Specific roles for CREB in neuronal development have been found by manipulating CREB function in vivo and in vitro (12.Herdegen T. Leah J.D. Brain Res. Brain Res. Rev. 1998; 28: 370-490Crossref PubMed Scopus (1155) Google Scholar). Bruton's tyrosine kinase (BTK) family kinases are members of a group of nonreceptor tyrosine kinases, which include BTK, Tec, and Bmx. These kinases have a canonical pleckstrin homology (PH) domain at the amino terminus, followed by cysteine-rich and proline-rich regions, which together have been termed the Tec homology domain (13.Satterthwaite A.B. Witte O.N. Immunol. Rev. 2000; 175: 120-127Crossref PubMed Google Scholar). The proline-rich portion of the Tec homology domain mediates interactions with the Src homology-3 (SH3) domains in vitro and may stabilize regulatory interactions with Src-type kinases in vivo (14.Cheng G. Ye Z.S. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8152-8155Crossref PubMed Scopus (165) Google Scholar, 15.Yang W. Malek S.N. Desiderio S. J. Biol. Chem. 1995; 270: 20832-20840Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The expression of BTK is restricted to a subset of B cells and myeloid cell (16.Qiu Y. Kung H.J. Oncogene. 2000; 19: 5651-5661Crossref PubMed Scopus (187) Google Scholar) and is involved in signaling of B cell antigen receptor, mast cell FcϵR, interleukin-5 receptor, and interleukin-6 receptor. Mutations in BTK are associated with X-linked agammaglobulinemia in humans and X-linked immunodeficiency and an impaired B cell proliferation in mice. A large number of proteins interact with BTK and are phosphorylated in a BTK-dependent manner. These include BAP-135, pp70, WASP, c-Cbl, Sam-68, vav, and EWS (16.Qiu Y. Kung H.J. Oncogene. 2000; 19: 5651-5661Crossref PubMed Scopus (187) Google Scholar). Although these proteins are putative BTK regulators or effectors by virtue of their binding activity and/or BTK-dependent phosphorylation, there is no direct biological evidence that demonstrates their role in BTK signaling. Although BTK is expressed ubiquitously in nearly all cells of the hematopoietic lineages, only B cells have been shown to be vulnerable with respect to functional integrity. In addition, the molecular mechanisms underlying BTK activation in other cells, including neuronal cells, are not well understood. Interestingly, one of the genes in the breakpoint region of 7q11.23 in Williams-Beuren syndrome, a neurodevelopmental disorder with multisystemic manifestations, which is caused by a heterozygous deletion in 7q11.23, encodes BAP-135, a phosphorylation target of BTK (17.Perez Jurado L.A. Wang Y.K. Peoples R. Coloma A. Cruces J. Francke U. Hum. Mol. Genet. 1998; 7: 325-334Crossref PubMed Scopus (149) Google Scholar). This finding suggests that the signal transduction mediated via BTK activation appears to be important for normal neural development. Recently it was shown that a small pool of BTK could translocate to the nucleus, although it continues to be present predominantly in the cytoplasm (18.Mohamed A.J. Vargas L. Nore B.F. Backesjo C.M. Christensson B. Smith C.I. J. Biol. Chem. 2000; 275: 40614-40619Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). BTK can also translocate to the plasma membrane upon growth factor stimulation (19.Nore B.F. Vargas L. Mohamed A.J. Branden L.J. Backesjo C.M. Islam T.C. Mattsson P.T. Hultenby K. Christensson B. Smith C.I. Eur. J. Immunol. 2000; 30: 145-154Crossref PubMed Scopus (95) Google Scholar). To identify the upstream signal transduction pathways in which CREB functions, we sought to identify proteins that interact specifically with and directly phosphorylate CREB in vivo. In the present study, we demonstrate that CREB is a substrate for BTK phosphorylation. Interestingly, active BTK directly phosphorylates CREB on Ser-133, suggesting that BTK could be a member of dual specificity protein kinases that are capable of phosphorylating both serine/threonine and tyrosine residues. Mutations of BTK which impair its activation or the suppression of endogenous BTK by siRNA duplexes lead to the abolishment of BTK-dependent phosphorylation of CREB and to a significant inhibition of neurite outgrowth induced by neurogenic growth factor. These findings suggest that CREB lies downstream of BTK in a signaling pathway started by neurogenic bFGF, and CREB activation via BTK is important for neuronal differentiation in central nervous system hippocampal progenitor cells. Materials—Peroxidase-conjugated anti-rabbit and anti-mouse IgGs were purchased from Zymed Laboratories, Inc. (San Francisco). Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), and LipofectAMINE PLUS reagent from were from Invitrogen. Protein A-Sepharose was from Amersham Biosciences. Anti-CREB and anti-phospho-CREB antibodies were from PerkinElmer Life Sciences and Upstate Biotechnology (Lake Placid, NY), respectively. Anti-rabbit polyclonal anti-BTK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). ECL reagents and [γ-32P]ATP were from PerkinElmer Life Sciences. Selective Src kinase inhibitor PP1 was from A. G. Scientific, Inc. (San Diego). The synthetic yeast dropout medium (SD/-T, SD/-L, SD/-HLT) and yeast extract peptone dextrose containing adenine were from Bio 101, Inc. (Vista, CA). 3-Amino-1,2,4-triazole and 5-bromo-4-chloro-3-indolyl-d-galactoside were from Sigma and Promega, respectively. The human fetal brain cDNA library was from Clontech. Human bFGF was from Sigma, and the luciferase assay kit was purchased from Promega. pVP16-CREB and pVP16-CREB S133A were gifts from K. Saeki (Research Institute in International Medical Center, Tokyo, Japan). The hemagglutinin-tagged wild type or kinase-inactive BTK mutants (K430E, in which the Lys-430 in the kinase domain of BTK was mutated to Glu) were gifts from A. L. Roy (Tufts University School of Medicine, Boston). The double tyrosine-mutated BTK construct (pEGFP-BTK-2MT-Y223A/Y551F, in which Tyr-223 in the SH3 domain of BTK and Tyr-551 in the kinase domain were replaced by Ala and Phe, respectively) was generously provided by A. J. Mohamed (Karolinska Institute, Huddinge, Sweden). A eukaryotic expression vector encoding constitutive active BTK, pSR-MSVTK-caBTK, in which Glu-41 residue in the PH domain was replaced by Lys, was kindly provided by O. N. Witte (University of California, Los Angeles). A prokaryotic expression plasmid encoding bacterially recombinant GST-caBTK was constructed by inserting the NotI fragment of the caBTK sequence digested from pSR-MSVTK-caBTK into pGEX4T1 vector (Amersham Biosciences). The plasmids encoding v-Src (pMvSrc) and mutant Src (pcSrc295Arg) were gifts from D. Foster (Hunter College of the City University of New York). Yeast Two-hybrid Assay—The bait vector for yeast two-hybrid assay was constructed by subcloning the mutant CREB cDNA, in which RRPSY (from amino acids 130–134) was replaced by RRSLY, into pHybTrp/Zeo. Human fetal cDNA library subcloned into prey vector (pACT2) was purchased from Clontech. All yeast two-hybrid screening protocols were performed as described previously (20.Yang E.J. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 39819-39824Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Sequencing of the inserts in positive library plasmids was performed using an automatic DNA sequencer (ALF express, Amersham Biosciences). Cell Culture—Immortalized hippocampal neuronal cells (H19-7) were grown on poly-l-lysine-coated cell culture dishes in Dulbecco's modified Eagle's medium containing 10% FBS and 100 units/ml penicillin-streptomycin (21.Eves E.M. Tucker M.S. Roback J.D. Downen M. Rosner M.R. Wainer B.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4373-4377Crossref PubMed Scopus (139) Google Scholar). The cells were maintained at 33 °C under 5% CO2 and stimulated with 10 ng/ml neurogenic growth factor, bFGF, to induce neuronal differentiation, after being treated with serum-free N2 medium for 2 days, as described previously (22.Chung K.C. Gomes I. Wang D. Lau L.F. Rosner M.R. Mol. Cell. Biol. 1998; 18: 2272-2281Crossref PubMed Scopus (63) Google Scholar). To prepare the cell lysates, cells were rinsed twice with ice-cold phosphate-buffered saline and then solubilized in lysis buffer (20 mm Tris, pH 7.9, containing 1.0% Triton X-100, 1 mm Na3VO4, 137 mm NaCl, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 mm sodium orthovanadate, 1 mm EGTA, 10 mm NaF, 1 mm tetrasodium pyrophosphate, 5 mm Na2EDTA, 10% glycerol, 1 mm β-glycerophosphate, 0.1 g/ml p-nitrophenyl phosphate, and 0.2 mm phenylmethylsulfonyl fluoride). The cells were removed by scraping, and the supernatants were collected after centrifugation for 15 min at 14,000 × g at 4 °C. Primary cortical neurons were obtained from rat fetal brain on embryonic day 15 and triturated. Dissociated cells were plated in 24-well culture plates coated with 100 mg/ml poly-d-lysine and 4 mg/ml laminin and maintained in Eagle's minimal essential medium (Eagle's salts, glutamine-free) supplemented with 21 mm glucose, 5% FBS (Biofluid), 5% horse serum, and 2 mm glutamine, at 37 °C under a humidified atmosphere of 95% air plus 5% CO2. Plating density was adjusted to 105 cells/culture well. After 2 days, the culture medium was replaced with growth medium identical to plating medium, but lacking fetal serum, and then fed twice a week. The U937 cell line was cultured in RPMI 1640 supplemented with 10% FBS and kept in a humidified incubator at 37 °C in 5% CO2 plus 95% air. SH-SY5Y cell line was grown on Dulbecco's modified Eagle's medium containing 10% FBS and 100 units/ml penicillin-streptomycin. The cells were maintained in a humidified incubator at 37 °C under 5% CO2 condition. Immunohistochemistry—Adult mice were perfused with 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4. Brains were removed and postfixed in the same buffer for 24 h at 4 °C. Thereafter, they were cryoprotected in 30% sucrose, frozen on dry ice, and sectioned on a cryostat. Serial coronal or sagittal sections (40 μm thick) were collected in a cryoprotectant solution (30% glycerol, 30% ethylene glycol, 40% 0.1 m phosphate buffer, pH 7.4). Immunohistochemistry was performed by using polyclonal anti-BTK antibodies and developed using avidin-biotin technique (Elite ABC kit, Vector Laboratories). DNA Transfection and Luciferase Reporter Assay—H19-7 cells were plated at a density of 2 × 106 cells/well in 100-mm dishes, were transfected when 70∼90% confluent with suitable plasmid constructs using LipofectAMINE PLUS reagent according to the manufacturer's instructions. The luciferase reporter construct, pCRE-TK-Luc, was transiently cotransfected with the kinase-inactive mutant BTK kinases (Y223A/Y551F, R28C, or K430E), and luciferase activity was measured using a luciferase assay kit (Promega) and a luminometer (EG & G Berhold, Germany). The CRE-lacking TK promoter construct (pTK-Luc) was used as a negative control throughout. Microinjection of H19-7 Cells—Around 4.5 × 104 cells were seeded to poly-l-lysine-coated coverslips placed on 6-well plate and grown at 33 °C. After the cells grew to the desired densities, anti-rabbit polyclonal anti-BTK antibody or anti-mouse control IgG (0.5 mg/ml) was microinjected with the injection buffer (50 mm HEPES, pH 7.4, 40 mm NaCl) containing 0.5% dextran rhodamine into the cell nuclei. Microinjection was done with an Eppendorf Micromanipulator 5171, Microinjector FemtoJet, and Olympus DP50 microscope. The cells were recovered in 10% FBS at 33 °C overnight before being shifted to 39 °C in N2 medium and were treated with 10 ng/ml bFGF. The cells containing dextran rhodamine were examined by immunofluorescence microscopy. Immunoprecipitation—1 μg of polyclonal anti-BTK antibody was incubated with 600 μg of cell extract prepared in lysis buffer overnight at 4 °C. 40 μl of a 1:1 suspension of protein A-Sepharose beads was added and incubated for 2 h at 4 °C with gentle rotation. The beads were pelleted and washed extensively with cell lysis buffer. Bound proteins were dissociated by boiling in SDS-PAGE sample buffer, and whole protein samples were separated on SDS-polyacrylamide gel. Western Blot Analysis—Whole-cell lysates were separated by electrophoresis through a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Japan). The membrane was blocked in TBST buffer containing 20 mm Tris, pH 7.6, 137 mm NaCl, 0.05% Tween 20, and 3% nonfat dry milk for 3 h, and then incubated overnight at 4 °C in 3% nonfat dry milk containing anti-phospho-CREB, anti-CREB, or anti-BTK antibodies (Santa Cruz Biotechnology). The membrane was washed several times in TBST and then incubated with secondary IgG-coupled horseradish peroxidase antibody (Zymed Laboratories Inc.). After 60 min, blots were washed several times with TBST and visualized by enhanced chemiluminescence. Analysis of bFGF-induced Neuronal Differentiation in H19-7 Cells— H19-7 cells were grown on poly-l-lycine-coated 6-well dishes to reach 70∼90% confluence. The cells were transfected with 1 μg of mammalian constructs encoding wild type or dominant negative BTK mutants, such as kinase-inactive K430E or Y223A/Y551F mutant of BTK using LipofectAMINE reagent. The next day the cells were switched to N2 medium and cultured at 39 °C for 2 days. They were then treated with 10 ng/ml bFGF for 48 h, and any morphological changes were noted. Differentiated cells were defined as cells with a round and refractory cell body and at least one neurite of length exceeding the diameter of the cell body. In Vitro BTK Kinase Assay—H19-7 cells grown in serum-free N2 medium for 2 days at 39 °C were treated with bFGF. The cells were harvested and lysed in lysis buffer, and 600 μg of the proteins so obtained were incubated with polyclonal BTK antibody overnight at 4 °C. The immunocomplexes were then added to 40 μl of a 1:1 suspension of protein A-Sepharose. After washing the samples three times in lysis buffer, kinase reactions were carried out at 30 °C for 60 min in 20 μl of kinase buffer containing 20 mm HEPES, pH 7.2, 5 mm MnCl2, 200 μm sodium orthovanadate, 5 μg of acid-treated enolase, 10 μm ATP, 5 μCi of [γ-32P]ATP, and 5 μg of wild type GST-CREB or mutant GST-CREB S133A as a substrate. The reactions were stopped by adding SDS-sample buffer and analyzed by SDS-PAGE and autoradiography. Assay of Bacterially Recombinant Fusion GST-BTK Protein with Constitutive Active Kinase Activity—The Sepharose-4B beads prebound to bacterially expressed GST-BTK with a constitutive active kinase activity were prepared by using Bulk GST purification module (Amersham Biosciences). 30 μl of beads were suspended in 200 μl of binding buffer (25 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 30 mm MgCl2, 40 mm NaCl, 0.5% Nonidet P-40) and mixed with 300 μg of either wild type GST-CREB or mutant GST-CREB S133A proteins for 2 h at 4 °C. After the bead pellet was washed twice with kinase buffer containing 20 mm HEPES, pH 7.2, 5 mm MnCl2, 200 μm sodium orthovanadate, and 10 μm ATP, the pellet was resuspended in 20 μl of kinase buffer, and kinase reactions were carried out by adding 5 μCi of [γ-32P]ATP at 30 °C for 60 min. The reactions were terminated by adding of SDS-sample buffer, and the phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. Proteomic Analysis of BTK Immunocomplexes—The total cell lysates were immunoprecipitated with a polyclonal anti-BTK IgG. After the beads were washed three times with lysis buffer, bound proteins were dissociated by boiling of the beads in SDS-PAGE sample buffer, and the eluted whole proteins were separated on 8% SDS-polyacrylamide gel. Next the gel was stained with Coomassie Blue R-250 dye followed by destaining overnight, and the individual protein band was digested. Each gel piece was then immersed in 100 μl of acetonitrile, dried under vacuum centrifugation for 10 min, rehydrated in trypsin buffer containing 1 μg/μl trypsin plus 50 mm ammonium bicarbonate, immersed in 100 μl of 50 mm ammonium bicarbonate, pH 8.0, incubated for 18 h at 37 °C, and analyzed by MALDI-TOF MS. In Vitro In-gel Kinase Assay—An 8.0% SDS-polyacrylamide gel was prepared in the presence of 50 μg/ml bacterial recombinant wild type GST-CREB or mutant GST-CREB-S113A proteins as a phosphorylation substrate. The total cell extracts were prepared after the stimulation of H19-7 cells with bFGF and applied to the gel. All gel renaturation and phosphorylation protocols were performed as described previously (22.Chung K.C. Gomes I. Wang D. Lau L.F. Rosner M.R. Mol. Cell. Biol. 1998; 18: 2272-2281Crossref PubMed Scopus (63) Google Scholar). Construction of siRNA Duplexes and Transfection—The siRNA duplexes for BTK were designed as described elsewhere (50.Heinonen J.E. Edvard Smith C.I. Nore B.F. FEBS Lett. 2002; 527: 274-278Crossref PubMed Scopus (62) Google Scholar). The cDNA-targeted region and the sequence of the siRNA duplexes for BTK are as follows: targeted region (cDNA), 895-GGGAAAGAAGGAGGTTTCA-913; sense siRNA, 5′-GGGAAAGAAGGAGGUUU CAUU-3′; antisense siRNA, 3′-UUCCCUUUCUUCCUCCAAAGU-5′. The siRNA duplexes of BTK were transfected by using LipofectAMINE PLUS reagent according to the manufacturer's instructions. Identification of Novel CREB Kinase(s) Activated during Neuronal Differentiation in Immortalized Hippocampal Progenitor Cells—The conditionally immortalized hippocampal cell line (H19-7) was generated by transducing temperature-sensitive SV40 large T antigen into rat embryonic day 17 hippocampal cells (21.Eves E.M. Tucker M.S. Roback J.D. Downen M. Rosner M.R. Wainer B.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4373-4377Crossref PubMed Scopus (139) Google Scholar). Central nervous system progenitor H19-7 cells have the ability to differentiate terminally into neuronal cells, at a nonpermissive temperature (39 °C), in the presence of several agents, such as bFGF. Differentiating H19-7 cells express several neuronal markers, including neurite outgrowth and neurofilament proteins. In an earlier study we showed that CREB phosphorylation and subsequent CRE-mediated gene transcription play an important role during bFGF-induced neuronal differentiation in H19-7 cells (23.Sung J.Y. Shin S.W. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 13858-13866Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Interestingly, CREB phosphorylation is not likely to be mediated by any of the previously known signaling pathways such as the MAPK, protein kinase A, protein kinase C, p70S6K, or Ca2+/calmodulin-dependent protein kinase pathways. These findings suggest that the activation of a novel protein kinasesignaling pathway is required to induce bFGF-responsiveness (23.Sung J.Y. Shin S.W. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 13858-13866Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Furthermore, the activation of two previously unreported CREB kinases of ∼76 and 120 kDa by bFGF was observed by in vitro in-gel kinase assay with recombinant GST-CREB fusion proteins as substrates. CREB phosphorylation on Ser-133 is considered to be a critical step in CREB transcriptional activation. Moreover, the modification of either the catalytic or the regulatory domain of the transcription factor is frequently used to enhance and to stabilize complex formation with its kinase in the yeast two-hybrid assay (24.Prigent S.A. Methods Mol. Biol. 2001; 124: 251-270PubMed Google Scholar, 25.Radhakrishnan I. Pérez-Alvarado G.C. Dyson H.J. Wright P.E. FEBS Lett. 1998; 430: 317-322Crossref PubMed Scopus (127) Google Scholar, 26.Parker D. Ferreri K. Nakajima T. LaMorte V.J. Evans R. Koerber S.C. Hoeger C. Montminy M.R. Mol. Cell. Biol. 1996; 16: 694-703Crossref PubMed Scopus (334) Google Scholar). The same assay was performed to identify the upstream signal transduction pathways leading to CREB phosphorylation and to isolate the novel CREB kinase(s), using CREB as bait, in which the critically regulatory Pro-132 and Ser-133 residues were changed to Ser-132 and Leu-133. As a result of screening of human fetal brain cDNA library, many CREB-interacting proteins were identified, including histone deacetylase and Dyrk1A (20.Yang E.J. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 39819-39824Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). In addition, it was found that BTK specifically interacts with CREB in yeast cells (data not shown). Because the molecular size of BTK (77 kDa) is comparable with that of one of the novel CREB kinases (23.Sung J.Y. Shin S.W. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 13858-13866Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and BTK plays an integral role in the differentiation and signal transduction directed by antigen-receptor in B cells, its functional role during neuronal differentiation was further examined. Specific Binding between Active CREB and BTK during Neuronal Differentiation in H19-7 Cells—First, we examined whether BTK is expressed in various types of mammalian primary and transformed neuronal cells. As shown in Fig. 1A, immunohistochemical analysis has shown that significant levels of BTK are expressed in a variety of mice central nervous system regions, such as neocortex, cerebral cortex, cerebellum, and hippocampus. Furthermore, considerable levels of BTK protein are normally present in transformed neuronal cells. These includes human neuroblastoma SH-SY5Y cell line, immortalized rat embryonic hippocampal H19-7 cells, and rat pheochromocytoma PC12 cell line, compared with that in several immune B cells as a control (Fig. 1B). We also observed that BTK is enriched in the cell extracts from rat whole brain and cerebellum (Fig. 1B). These observations suggest that, outside of hematopoietic cells, BTK could play a role during cell differentiation and signal transduction in neuronal cells. To confirm the previous finding that bFGF exerts its stimulatory effect on the activation of CREB (23.Sung J.Y. Shin S.W. Ahn Y.S. Chung K.C. J. Biol. Chem. 2001; 276: 13858-13866Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), Western blot analysis was performed using an antibody specific for the Ser-133-phosphorylated form of CREB. During the differentiation of H19-7 cells by bFGF, the Ser-133 residue in the CREB protein was phosphorylated, but endogenous CREB levels were not markedly changed (Fig. 1C). Then we examined whether BTK specifically binds to CREB in hippocampal H19-7 cells. The H19-7 cell extracts obtained after neurogenic bFGF stimulation were immunoprecipitated using anti-BTK antibodies and then blotted using anti-phospho-CREB antibodies. As shown in Fig. 1C, the expression of BTK is not markedly changed by bFGF. However, the addition of bFGF leads to a marked increase in the specific binding between BTK and phospho-CREB. Furthermore, when the cell lysates were immunoprecipitated with anti-phospho-CREB antibodies and analyzed by Western blot using anti-BTK IgG, it was found that the addition of bFGF resulted in specific binding between BTK and phospho-CREB (Fig. 1C). These results suggest that BTK interacts with active CREB in a speci" @default.
• W2039659863 created "2016-06-24" @default.
• W2039659863 creator A5017641872 @default.
• W2039659863 creator A5025236447 @default.
• W2039659863 creator A5050119772 @default.
• W2039659863 date "2004-01-01" @default.
• W2039659863 modified "2023-10-16" @default.
• W2039659863 title "Bruton's Tyrosine Kinase Phosphorylates cAMP-responsive Element-binding Protein at Serine 133 during Neuronal Differentiation in Immortalized Hippocampal Progenitor Cells" @default.
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