Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 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-Calpha) 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-Calpha in vivo. In COS-7 cells cotransfected with PKA-Calpha 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 (S(64) and S(211)) 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 Col2a1 chondrocyte-specific enhancer; similarly, cotransfection of PKA-Calpha increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Calpha 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.

FIG. 1

PKA-Cα interacts with SOX9 in yeast cells. (A) Bait plasmid used in the SRS screening. The full-length coding sequence of SOX9 was cloned into pADNS in frame with and carboxy-terminal to the SOS coding sequence. Six glycine residues were inserted between the SOX9 and SOS sequences. (B) Complementarity of the cdc25-2 temperature-sensitive mutation through specific interactions of SOX9 with PKA-Cα. The plasmid isolated from a yeast colony, which was positive upon primary library screening and encoded PKA-Cα, was retransformed into the cdc25-2 mutant strain together with either empty pADNS, pADNS-p110-SOS, or pADNS-SOS-SOX9. Four independent colonies were isolated for each plasmid combination, grown at 25°C, replica plated onto galactose and glucose (control) plates, and grown at 37°C. Only the colonies that expressed SOS-SOX9 and PKA-Cα grew on the galactose plates.

FIG. 2

Mutations of the two PKA phosphorylation sites of SOX9. (A) The amino acid sequence of human SOX9 is shown from amino acids 56 to 216, with the HMG domain highlighted in bold and the two PKA phosphorylation consensus sites shown in boxes. (B) Serine 64 and serine 211 in the PKA phosphorylation consensus sites were replaced with alanine in SOX9 mutant m1 and SOX9 mutant m2, respectively. SOX9 mutant m1+2 contained alanine substitutions at both sites.

FIG. 3

Phosphorylation of SOX9 by PKA in vitro. (A) Time course. Bacterially expressed SOX9 was incubated with PKA-Cα in the presence of [γ-³²P]ATP for 0 to 30 min, as indicated, before PKI was added to terminate the reactions. Reaction mixtures were separated by SDS-PAGE, and the gels were autoradiographed. A time-dependent increase in phosphorylated protein was obtained at the characteristic migration level of SOX9. Two faster-migrating products of PKA-Cα phosphorylation were detected that presumably correspond to incomplete or degraded recombinant SOX9 polypeptides (not shown). (B) Specific phosphorylation of sites 1 and 2. Bacterially expressed wild-type (wt) or double-mutant (m1+2) SOX9 proteins were incubated with (+) or without (−) PKA-Cα and PKI for 30 min, as indicated above the lanes. Phosphorylation of SOX9 was determined as described for panel A. A Western blot of wild-type and m1+2 mutant SOX9 proteins shows that the same amount of each SOX9 protein species was present in all reactions.

FIG. 4

Phosphorylation of Sox9 in intact cells. (A) Endogenous Sox9 is phosphorylated in RCS cells. RCS cells in culture were incubated with ³²P-labeled inorganic phosphate for 4 h. Sox9 was immunoprecipitated from cell lysates with SOX9 antibody (Ab) and visualized by autoradiography after SDS-PAGE (lane SOX9 Ab). A unique ³²P-labeled band migrated with the characteristic 68-kDa molecular mass of SOX9. The specificity of the immunoprecipitation was assessed with SOX9 preimmune (Pre-immu.) serum. (B) PKA-Cα phosphorylates SOX9 at sites 1 and 2 in COS-7 cells. COS-7 cells were transfected with plasmids expressing wild-type SOX9 (wt), double-mutant SOX9 (m1+2), and PKA-Cα, as indicated. Cells were incubated with ³²P, and lysates were immunoprecipitated with SOX9 antibody. Autoradiographs of phosphorylated SOX9 proteins recovered after immunoprecipitation and SDS-PAGE are shown in the top panel. PKA increased the level of phosphorylation of wild-type SOX9 but not that of m1+2 mutant SOX9. A Western blot showing the amount of SOX9 protein in each sample after immunoprecipitation is presented in the bottom panel.

FIG. 5

PKA phosphorylation of SOX9 does not affect its nuclear localization. COS-7 cells were transfected with expression plasmids for wild-type SOX9, m1+2 mutant SOX9, and PKA-Cα, as indicated. The top panels (A to D) show the nuclear localization of the wild-type and m1+2 mutant SOX9 proteins (yellow signal) visualized by immunofluorescence with SOX9 antibodies. The lower panels represent the same fields in which cell nuclei were stained with DAPI.

FIG. 6

PKA and cAMP stimulate the activity of chondrocyte-specific Col2a1 enhancers. (A) 8-Bromo-cAMP increased the activity of the 48-bp Col2a1 enhancer in RCS cells. A luciferase reporter plasmid containing no (p89Luc) or four copies of a 48-bp chondrocyte-specific enhancer element (4x48-p89Luc) was transfected into RCS cells, and 4 h later, 1 mM 8-bromo-cAMP was added (+cAMP) or not (−) to the culture medium for 8 h. Promoter activities were shown as the average plus standard deviation of triplicate transfections for A and B. (B) PKA-Cα increases the SOX9-dependent activation of the 48-bp Col2a1 enhancer in 10T1/2 fibroblasts. Expression plasmids for SOX9 and PKA-Cα were either transfected individually or cotransfected in a 1:4 ratio for a total of 16 h. Where indicated, the PKA inhibitor H8 was added to the medium for 12 h beginning 4 h after the start of transfection. (C) Cotransfected PKA-Cα increases the SOX9-dependent activation of the 100-bp Col2a1 enhancer in 10T1/2 fibroblasts. A luciferase reporter plasmid containing two copies of the 100-bp enhancer was cotransfected without (−) or with the expression plasmids for SOX9 and PKA-Cα into 10T1/2 fibroblasts for a total of 16 h. Eight hours after the start of transfection, 1 mM 8-bromo-cAMP was added as indicated, and the mixture was incubated for an additional 8 h. Solid and hatched columns show the results of separate duplicate transfections.

FIG. 7

Mutations in the PKA phosphorylation sites of SOX9 decrease the ability of PKA to enhance SOX9 activation of the Col2a1 enhancer. COS-7 cells were transfected with the 4x48-p89Luc reporter construct and plasmids encoding wild-type SOX9 (lanes 3 and 7) or one of the SOX9 mutants (m1, lanes 4 and 8; m2, lanes 5 and 9; m1+2, lanes 6 and 10), and PKA-Cα (lanes 2 and 7 to 10). Lane 1 is a transfection with the 4x48-p89Luc reporter plasmid alone. Promoter activity was determined 12 h later and is shown as the average plus standard deviation of triplicate transfections. A Western blot of cell lysates was probed with SOX9 antibody to show the relative amounts of each of the SOX9 proteins. In cells cotransfected with PKA-Cα, the levels of SOX9 were 1.5 to 1.8 times higher than in cells not cotransfected with PKA-Cα. Promoter activities were normalized relative to the levels of SOX9 protein.

FIG. 8

Phosphorylation of SOX9 by PKA increases its efficiency of binding to the 18-bp Col2a1 enhancer element. Bacterially expressed and purified wild-type (wt) (A) or m1+2 mutant (B) SOX9 protein was incubated in vitro with PKA-Cα. Nonphosphorylated and phosphorylated proteins were then tested in an EMSA with the ³²P-labeled 18-bp Col2a1 enhancer element as a probe. A 100-fold excess of unlabeled 18-bp oligonucleotide competitor and PKI were added to the phosphorylation reactions, as indicated. The positions of wild-type and m1+2 mutant SOX9 complexes with the 18-bp probe are marked.

FIG. 9

Phosphorylation of Sox9 at S₂₁₁ in intact cells. (A) Recombinant SOX9 protein made in E. coli and extracts from COS-7 cells that were transfected with plasmids expressing wild-type (wt) SOX9 or mutant SOX9 proteins and a plasmid expressing PKA-Cα, as indicated, were resolved by SDS–10% PAGE followed by Western blotting with SOX9.P antibody (Ab) at a dilution of 1:1,000. The antibodies were stripped off the blots, and the membranes were reprobed with another SOX9 antibody as described previously (20). In A and B, the 68-kDa mobility of Sox9 is indicated by an arrow. (B) Cell extracts from RCS cells, untreated or treated with two different concentrations of 8-bromo-cAMP for 8 h, were used for Western blotting.

FIG. 10

Sox9 is phosphorylated at S₂₁₁ in the prehypertrophic zone of the growth plate in vivo. (A) Immunohistochemistry of a hind limb section of E 16.5 mouse embryo with SOX9 antibodies. Sox9 is evenly distributed in the cells of the resting, proliferative (P), and prehypertrophic (Pre-hy) zones of the growth plate. No Sox9 protein was observed in the hypertrophic (hy) zone. (B) Immunohistochemistry of a parallel section with SOX9.P antibodies. Sox9 phosphorylated at S₂₁₁ was present in the prehypertrophic zone of the growth plate. Bars, 50 μm.