REGULATION OF CREB PHOSPHORYLATION BY camp AND CA 2+ IN PAROTID ACINAR CELLS
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1 Vol. 43, No. 3, October 1997 BIOCHEMISTRY end MOLECULAR BIOLOGY INTERNATIONAL Pages REGULATION OF CREB PHOSPHORYLATION BY camp AND CA 2+ IN PAROTID ACINAR CELLS Taishin Takuma TM, Yoshifumi Tajima 2, and Tokuro Ichida ~ ~Department of Oral Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido , Japan 2Department of Oral Pathology, School of Dentistry, Meikai University, Sakado, Saitama , Japan Received July 24, 1997 Summary: Since various secretory stimuli regulate not only secretion but also protein, RNA, and DNA syntheses in salivary glands, we evaluated the effect of secretory stimuli on the phosphorylation state of CREB (camp response elementbinding protein). Isoproterenol, forskolin, and CPS-cAMP markedly stimulated the phosphorylation of CREB in parotid acinar cells, and PKA inhibitors H-8 and H-89 dose-dependently inhibited it. In contrast, carbachol (CCH) and A23187 decreased CREB phosphorylation, but CCH did not decrease it in the absence of extracellular Ca 2+. Although protein phosphatase inhibitor calyculin A alone markedly increased the phosphorylation, it could not prevent CCH-induced dephosphorylation of CREB. CaM kinase IV, a putative protein kinase for CREB in response to Ca 2 elevation, was undetectable in parotid acinar cells. Key words: CREB, PKA, Ca 2~, CaM kinase IV, Parotid acinar cell. Introduction The transcription factor CREB is well established to be activated by phosphorylation at serine 133 (1,2). The phosphorylated CREB is associated with CREB-binding protein (CBP) and enhances transcription of the gene that has a camp response element (CRE)in its promotion site (3). CREB is phosphorylated by the catalytic subunit of PKA, which kinase is translocated from the cytosol to the nucleus in response to an increase in cellular camp (4). In addition to PKA, many other protein kinases, including PKC, Ca2+/calmodulin-dependent protein kinase (CaM kinase) I, II and IV, can phosphorylate CREB (5-8) but the identity of the enzyme that directly catalyzes the phosphorylation in vivo has not yet been fully established. Although CaM kinase II phosphorylates CREB, it rather inhibits the transcriptional activity of "To whom correspondence should be addressed: Tel.: (81) Fax.: (81) takuma@hucc.hokudai.ac.jp /97/ /0 Copyright by Academic Press Australia. All rights of reproduction in any farm reserved
2 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL CREB by an additional phosphorylation of serine-142 in a negative regulation site (9). Transfection with expression vectors for constitutively active forms of both CaM kinase I and IV can activate CREB (10). Among these CaM-dependent enzymes, however, only CaM kinase IV is localized in nuclei where CREB is also located (11-13). The salivary gland has various receptors for extracellular signaling molecules and elicits a wide range of responses, including secretion, protein synthesis, and cell proliferation. Amoung signaling molecules, isoproterenol (ISO), a typical 13-adrenergic agonist, is the strongest known stimulant for salivary protein secretion, and its chronic administration induces hypertrophy and hyperplasia in salivary glands in vivo. ISO and camp analogues also induce rapid transcription of various mrnas, including those of c-los (14), amylase (15) and proline-rich proteins (16,17) in cultured parotid acini. On the other hand, carbachol (CCH), a typical muscarinic cholinergic agonist induces fluid and protein secretion through mobilization of intracellular Ca 2. and activation of PKC (18,19). However, CCH strongly inhibits both protein and RNA syntheses (20). Although CREB is highly possible to be involved in the regulatory mechanism of these processes, so far no data is available concerning the phosphorylation state of CREB during stimulation with these agents. Thus, in this study, we evaluated the effect of various stimuli on CREB phosphorylation in rat parotid acinar cells. Materials and Methods Materials :FK506 was a gift from Fujisawa (Osaka, Japan). Rabbit polyclonal antibodies to CREB and phosphorylated CREB at serine-133 were purchased from New England Biolabs (Beverly, MA, USA). Goat polyclonal antibody to CaM kinase IV (M-20) was from Santa Cruz Biotechnology (Santa Cruz, CA). H-8 and H-89 were obtained from Seikagaku Kogyo (Tokyo). Calyculin A was from Wako (Osaka); polyvinylidene difluoride (PVDF) membrane (Immobilon), from Millipore (Bedford, MA); enhanced chemiluminescence (ECL) kit, from Amersham (Little Chalfont, England); Block Ace, from Dainippon Seiyaku (Osaka, Japan); and Pefabloc SC, from Merck (Darmstadt, Germany). All other chemicals utilized were the highest grade commercially available. Detection of CREB and phospho-creb by immunoblotting :Rat parotid acinar cells (small acini) were prepared as described earlier (21,22). Suspensions of parotid acini were incubated with various stimulants at 37 ~ for 30 min in minimum essential medium buffered with 20 mm Na-Hepes (ph 7.4) containing 0.1% BSA (MEM-H) under continuous shaking (120 cycles per min). After incubation, the acini was pelleted, lysed with Laemmli cocktail and boiled for 5 rain. Proteins of parotid acini were resolved by SDS-PAGE on a 5-20% gradient gel (Atto, Tokyo) and transferred to a PVDF membrane at 100 ma per mini-gel (90 x 73 x 1 mm) for min with a semi-dry blotter using 0.1 M Tris M glycine buffer containing 5% methanol. The 564
3 Voi. 43, NO. 3, 1997 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL membrane was washed twice with water, blocked with Block Ace at room temperature for 1 h, and incubated with CREB or phospho-creb antibody (1: 1000) in PBS containing 0.05% Tween-20 and 20% Block Ace for overnight at 4 ~ as described previously (23,24). CREB and phospho-creb were then visualized by the ECL system. Densitometric analysis was carried out using NIH Image Results and Discussion The phosphorylation state of CREB in rat parotid acinar cells was monitored by immunoblotting with the specific antibody that recognizes phosphoserine-133 in the CREB. As shown in Fig. 1A, incubation of parotid acini with 1 #M ISO, 10 #M forskolin, or 1 mm CPS-cAMP for 30 min clearly enhanced CREB phosphorylation, indicating that an increase in camp phosphorylates CREB in parotid acini. Then we examined the effects of H-8 and H-89, selective inhibitors of PKA, on CREB phosphorylation (Fig. 1B). Preincubation with #M H-8 or tam H-89 for 30 min dose- dependently inhibited the phosphorylation. These experiments revealed that i) the inhibitory effect of H-8 at 400 #M was still incomplete and ii) the phosphorylation state of CREB at 100 tam H-89 was below the control level. These results are generally consistent with previous findings concerning the inhibitory effect of H-8 and H-89 on camp-mediated exocytosis from parotid acini, although 400 #M H-8 did not inhibit the exocytosis (21). Next we examined the effects of 10 #M CCH, 1 #M A23187, and 1 tam PMA (phorbol 12-myristate 13-acetate). CCH did not stimulate the phosphorytation of CREB, but rather decreased it slightly (Fig. 1A). Calcium ionophore A23187 decreased the phosphorylation more strongly than CCH. On the other hand, PMA slightly but reproducibly increased the band density, suggesting that PKC weakly phosphorylates CREB, as reported previously (8). As can be seen in Fig. 1, when phosphorylated CREB was increased or decreased by various treatments, the reactivity with total CREB antibody was also changed slightly. These results were reproducible in 4 independent experiments. It is presently unknown why A23187 markedly decreased the reactivity. In neuronal cells, CREB was rapidly phosphorylated in response to an increase in cellular Ca 2 within 1-3 min after stimulation and then gradually dephosphorylated during prolonged incubation for up to 90 min (13). Thus we examined much earlier the effect of CCH on CREB phosphorylation. As shown in Fig. 2A, however, the decrease 565
4 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL A CON ISO Frsk CPS CCH A231 PMA % % B CON ISO H8 H8 H89 H98 H % % Fig. 1 Effects of various secretory agonists and PKA inhibitors on CREB phosphorylation in parotid acinar cells. A: Parotid acini were incubated with 1 tam ISO, 10 ~M forskolin, 1 mm CPS-cAMP, 10 tam CCH, 1 ~M A23187, or 1 ~M PMA at 37 ~ for 30 min. B: Acini were preincubated with ~M H-8 or ~M H-89 for 30 min and further incubated for 30 min with 1 ~M ISO. After incubation, the acini were pelleted and lysed with Laemmli cocktail. The CREB phosphorylated at serine- 133 (upper lanes) and total CREB (lower lanes) were visualized and quantified by immunoblotting as described in the Experimental procedures. in CREB phosphorylation was detectable at 1 min and no increase in phosphorylation was detected at any time examined. Since CREB is reported to be dephosphorylated by either protein phosphatases t or 2A (25-28), we examined the effect of the protein phosphatase inhibitor calyculin A. As shown in Fig. 2B, calyculin A alone markedly increased the amount of phosphorylated CREB. However, CCH still decreased CREB phosphorylation even in the presence of calyculin A. In the absence of extracellular Ca 2+, however, CCH did not decrease the phosphorylation irrespective of the absence or presence of calyculin A, confirming that the dephosphorylation of CREB is Ca
5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL A CON CCH CCH CCH CCH % B % CON CCH Caly-A Caly-A +CCH ~ ~ ~ : m C CON CCH Caly-A Caly-A +CCH --Ca ~ ~ ~ % I't CON CCH Caly-A Caly-A I,.# +CCH +FK506 ~ ~ ~ ~ % Fig. 2 Effect of CCH on the phosphorylation of CREB. A: Time course of CREB dephosphorylation by CCH. Parotid acini were incubated with 10 tam CCH for 1-30 min. B: Effects of calyculin A and CCH. Acini were preincubated with 100 nm calyculin A or not for 5 min and then incubated with 10 ~tm CCH or not for an additional 30 min. C: Effects of catyculin A and CCH in Ca~+-free medium. Parotid acini were washed twice in Ca2"-free Hanks' balanced salt solution containing 1 mm EGTA and incubated as described for "B" in the same Ca~'-free medium. D: Effects of FK506, calyculin A, and CCH. Parotid acini were preincubated for 30 min with 1 tam FK506. The acini 'were then incubated for 35 min with 100 nm calyculin A and/or 10 tam CCH as described for "B" in fresh medium containing 1 tam FK506. The CREB phosphorylated at serine-133 was visualized by immunoblotting as described in Experimental procedures. dependent (Fig. 2C). Although FK506, an inhibitor of calcineurin, retarded the dephosphorylation of CREB in neuronal cells (13), 1 tam FK506 neither increased the phosphorylation nor prevented the dephosphorylation (Fig. 2D). Finally we examined whether or not parotid acinar cells have CaM kinase IV, a putative protein kinase for CREB phosphorylation in response to Ca ~+ signaling in vivo. As shown in Fig. 3, CaM kinase IV was clearly detected in the lysate of rat brain but not in parotid acinar cells. CaM kinase IV was unable to be detected in parotid acini even when a large amount of proteins was subjected to SDS-PAGE and the immunoblots were exposed for a longer time. 567
6 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL Par Bra Mr kda Fig. 3 Detection of CaM kinase IV in brain and parotid acinar cells. The rat brain was homogenized with ice-cold phosphate-buffered saline containing 0.2 mm Peferblock SC in a Teflon-glass homogenizer. The homogenate was mixed with Laemmli cocktail and boiled. Parotid acini were pelleted and lysed with Laemmli cocktail. Brain lysate (18 ~g protein) and parotid lysate (42 ~g protein) were resolved by SDS-PAGE, and CaM kinase IV was detected by immunoblotting. Bra, brain lysate; Par, lysate of parotid acinar cells. The present study clearly shows that in parotid acinar cells the phosphorylation state of serine-133 in CREB is sensitively regulated through camp-and Ca2*-mediated signalings toward opposite directions; i.e., camp increased CREB phosphorylation by PKA and Ca 2. decreased it by an as yet undefined mechanism. Increase in celluar Ca 2. did not stimulate CREB phosphorylation probably because parotid cells do not have sufficient CaM kinase IV, if any. Since calyculin A markedly enhanced CREB phosphorylation, protein phosphatase 1 and 2A must be mainly responsible for the dephosphorylation of CREB, as reported previously (25-28). However, Ca 2*- dependent dephosphorylation was not prevented even in the presence of calyculin A, suggesting that additional protein phosphatases are also involved in CREB dephosphorylation. However, preincubation with 1 tam FK506 alone for 30 min showed apparently no effect on the dephosphorylation, and FK506 plus calyculin A still incompletely inhibited the dephosphorylation. Thus, further study is necessary to define the role of calcineurin in the mechanism of CREB dephosphorylation (13). The change in phosphorylation states of CREB as described above is well compatible with the change in RNA synthesis found previously in salivary gland cells. Namely, agonists elevating cellular camp strongly induced transcription of various 568
7 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL mrnas, including those of c-fos (14), amylase (15), and proline-rich proteins (16,17). Cyclic AMP-mediated RNA synthesis was markedly inhibited by H-8 (29). PMA transiently but significantly increased RNA synthesis in parotid Iobules (30). Okadaic acid, a protein phosphatase inhibitor, also induced RNA synthesis and potentiated the effect of PMA (29). In contrast, CCH and A23187 markedly decreased the amylase mrna level in parotid glands (20). These results and our present findings collectively suggest that CREB plays a crucial role in the regulation of transcriptional activity in parotid acinar cells. Acknowledgement This study was supported in part by a grant-in-aid for special research projects from the Health Sciences University of Hokkaido. References 1. Montminy, M., Brindle, P., Arias, J., Ferreri, K. and Armstrong, R. (1996) Adv Pharmacol 36, Gonzalez, G.A. and Montminy, M.R (1989) Cell 59, Chrivia, J.C., Kwok, R.P., Lamb, N., Hagiwara, M., Montminy, M.R. and Goodman, R.H. (1993) Nature 365, Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, W., Tsien, R. and Montminy, M.R. (1993) Mol Cell Bio113, Sheng, M., Thompson, M.A. and Greenberg, M.E. (1991) Science 252, Dash, P.K., Karl, K.A., Colicos, MA., Prywes, R. and Kandel, E.R. (1991) Proc Natl Acad Sci U S A 88, Enslen, H., Sun, P., Brickey, D., Soderling, SH., Klamo, E. and Soderling, T.R. (1994) J Biol Chem 269, Xie, H. and Rothstein, T.L. (1995) J Immunol 154, Sun, P., Enslen, H., Myung, P.S. and Maurer, RA. (1994) Genes Dev 8, Sun, P., Lou, L. and Maurer, R.A. (1996) J Biol Chem 271, Matthews, R.P., Guthrie, C.R., Wailes, LM., Zhao, X., Means, A.R. and McKnight, G.S. (1994) Mol Cell Biol 14, Enslen, H., Tokumitsu, H. and Soderling, T.R. (1995) Biochem Biophys Res Commun 207, Bite, H., Deisseroth, K. and Tsien, R.W. (1996) Cell 87, Kousvelari, E., Louis, J.M., Huang, L.H. and Curran, T. (1988) Exp Cell Res 179, Kim, S.K., Jones, T.P. and Cuzzort, L.M. (1989) Arch Oral Biol 34, Mehansho, H., Clements, S., Sheares, B.T., Smith, S. and Carlson, D.M. (1985) J Biol Chem 260, Wright, P.S., Lenney, C. and Carlson, D.M. (1990) J Mol Endocrinol 4, Tojyo, Y., Matsui, S., Tanimura, A. and Matsumoto, Y. (1992) Biochim Biophys Acta 1134, Tojyo, Y., Tanimura, A., Matsui, S. and Matsumoto, Y. (1992) Cell Struct Funct 17,
8 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL 20. Liu, Y., Woon, P.Y., Lim, S.C., Jeyaseelan, K. and Thiyagarajah, P. (1995) Biochem J 306, Takuma, T. and Ichida, T. (1994) Febs Lett 3401, Takuma, T. and Ichida, T. (1994) J Biol Chem 269, Takuma, T., Ichida, T., Yokoyama, N, Tamura, S. and Obinata, T. (1996) J Biochem 120, 35-4T. 24. Takuma, T., Tagaya, M. and Ichida, T. (1997) Febs Lett 404, Hagiwara, M., Alberts, A., Brindle, P., Meinkoth, J., Feramisco, J., Deng, T., Karin, M., Shenolikar, S. and Montminy, M. (1992) Cell 70, Wadzinski, B.E., Wheat, W.H., Jaspers, S., Peruski, L., Jr., Lickteig, R.L., Johnson, G.L. and Klemm, D.J. (1993) Mol Cell Biol 13, Alberts, A.S., Montminy, M., Shenolikar, S. and Feramisco, J.R. (1994) Mol Cell Biol 14, Wheat, W.H., Roesler, W.J. and Klemm, D.J. (1994) Mol Cell Biol 14, Woon, P.Y., Jeyaseelan, K. and Thiyagarajah, P. (1993) Biochem Pharmacol 45, Woon, P.Y., Jeyaseelan, K. and Thiyagarajah, P. (1993) Arch Oral Biol 38,
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