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Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation.

Publication ,  Journal Article
Enslen, H; Sun, P; Brickey, D; Soderling, SH; Klamo, E; Soderling, TR
Published in: J Biol Chem
June 3, 1994

We have characterized Ca2+/calmodulin-dependent protein kinase IV (CaM kinase IV), expressed using the baculovirus/Sf9 cell system, to assess its potential role in Ca2+-dependent transcriptional regulation. CaM kinase IV was strongly inhibited in vitro by KN-62, a specific CaM kinase inhibitor which suppresses Ca2+-dependent transcription of several genes, so we tested whether CaM kinase IV could stimulate transcription. Co-transfection of COS-1 cells by cDNA for CaM kinase IV gave 3-fold stimulation of a reporter gene expression, whereas co-transfection with CaM kinase II gave no transcriptional stimulation. Since this transcriptional response was mediated by phosphorylation of cAMP responsive element-binding protein (CREB), we determined the kinetics and site specificities of CaM kinases IV and II for phosphorylating CREB in vitro. CaM kinases IV and II and cAMP kinase (protein kinase A) all had similar Km values for CREB (1-5 microns), but the Vmax of CaM kinase IV was 40-fold lower than those of CaM kinase II and protein kinase A. Although all three kinases phosphorylated Ser133 in CREB, CaM kinase II also gave equal phosphorylation of a second site which was not Ser98. The two CREB phosphorylation sites were separately 32P-labeled, and the abilities of protein phosphatases 1, 2A, and 2B (calcineurin) to dephosphorylate them were tested. Our results show that all three phosphatases could dephosphorylate both sites, and calcineurin was a stronger catalyst for dephosphorylating site 1 (Ser133) than for site 2. These results indicate that CaM kinase IV may be important in Ca2+-dependent transcriptional regulation through phosphorylation of Ser133 in CREB. The fact that CaM kinase II phosphorylates another site in addition to Ser133 in CREB raises the possibility that this second phosphorylation site may account for the suppressed phosphorylation site may account for the suppressed ability of CaM kinase II to enhance transcription through the CRE/CREB system. In addition multiple protein phosphatases, including calcineurin, may exert a modulatory effect on transcription depending on which site they dephosphorylate.

Duke Scholars

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 3, 1994

Volume

269

Issue

22

Start / End Page

15520 / 15527

Location

United States

Related Subject Headings

  • Transfection
  • Transcription, Genetic
  • Substrate Specificity
  • Restriction Mapping
  • Phosphorylation
  • Peptide Mapping
  • Peptide Fragments
  • Oligodeoxyribonucleotides
  • Moths
  • Molecular Sequence Data
 

Citation

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MLA
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Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., & Soderling, T. R. (1994). Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. J Biol Chem, 269(22), 15520–15527.
Enslen, H., P. Sun, D. Brickey, S. H. Soderling, E. Klamo, and T. R. Soderling. “Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation.J Biol Chem 269, no. 22 (June 3, 1994): 15520–27.
Enslen H, Sun P, Brickey D, Soderling SH, Klamo E, Soderling TR. Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. J Biol Chem. 1994 Jun 3;269(22):15520–7.
Enslen, H., et al. “Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation.J Biol Chem, vol. 269, no. 22, June 1994, pp. 15520–27.
Enslen H, Sun P, Brickey D, Soderling SH, Klamo E, Soderling TR. Characterization of Ca2+/calmodulin-dependent protein kinase IV. Role in transcriptional regulation. J Biol Chem. 1994 Jun 3;269(22):15520–15527.

Published In

J Biol Chem

ISSN

0021-9258

Publication Date

June 3, 1994

Volume

269

Issue

22

Start / End Page

15520 / 15527

Location

United States

Related Subject Headings

  • Transfection
  • Transcription, Genetic
  • Substrate Specificity
  • Restriction Mapping
  • Phosphorylation
  • Peptide Mapping
  • Peptide Fragments
  • Oligodeoxyribonucleotides
  • Moths
  • Molecular Sequence Data