Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate aCol2a1 Chondrocyte-Specific Enhancer

ABSTRACT Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Cα) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Cα in vivo. In COS-7 cells cotransfected with PKA-Cα and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S64 and S211) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1chondrocyte-specific enhancer; similarly, cotransfection of PKA-Cα increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Cα activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.

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Negative Regulation of Histone Deacetylase 8 Activity by Cyclic AMP-Dependent Protein Kinase A

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.765-773.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 765-773 ◽

Cited By ~ 99

Author(s):

Heehyoung Lee ◽

Natalie Rezai-Zadeh ◽

Edward Seto

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinase A ◽

Class I ◽

Dependent Protein Kinase ◽

Nonhistone Proteins ◽

Dependent Protein ◽

Class I Hdacs ◽

Kinase A

ABSTRACT Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from lysine residues of histone and nonhistone proteins. Recent studies suggest that they are key regulators of many cellular events, including cell proliferation and cancer development. Human class I HDACs possess homology to the yeast RPD3 protein and include HDAC1, HDAC2, HDAC3, and HDAC8. While HDAC1, HDAC2, and HDAC3 have been characterized extensively, almost nothing is known about HDAC8. Here we report that HDAC8 is phosphorylated by cyclic AMP-dependent protein kinase A (PKA) in vitro and in vivo. The PKA phosphoacceptor site of HDAC8 is Ser39, a nonconserved residue among class I HDACs. Mutation of Ser39 to Ala enhances the deacetylase activity of HDAC8. In contrast, mutation of Ser39 to Glu or induction of HDAC8 phosphorylation by forskolin, a potent activator of adenyl cyclase, decreases HDAC8's enzymatic activity. Remarkably, inhibition of HDAC8 activity by hyperphosphorylation leads to hyperacetylation of histones H3 and H4, suggesting that PKA-mediated phosphorylation of HDAC8 plays a central role in the overall acetylation status of histones.

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Impaired G-protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain is not accompanied by reduced cyclic-AMP-dependent protein kinase A activity

Brain Research ◽

10.1016/0006-8993(96)00724-x ◽

1996 ◽

Vol 737 (1-2) ◽

pp. 155-161 ◽

Cited By ~ 13

Author(s):

Willy L Bonkale ◽

Johan Fastbom ◽

Birgitta Wiehager ◽

Rivka Ravid ◽

Bengt Winblad ◽

...

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Protein Kinase A ◽

Adenylyl Cyclase ◽

G Protein ◽

Cyclase Activity ◽

Adenylyl Cyclase Activity ◽

Dependent Protein Kinase ◽

Dependent Protein ◽

Kinase A

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Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2790727 ◽

1991 ◽

Vol 279 (3) ◽

pp. 727-732 ◽

Cited By ~ 20

Author(s):

G B Sala-Newby ◽

A K Campbell

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Specific Activity ◽

Phosphorylation Site ◽

Live Cells ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Ph Optimum ◽

Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

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