Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor

Abstract

The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser(301) and Ser(319) within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERK-dependent phosphorylation at Ser(319), mass spectroscopy identified two other ERK-phosphorylated sites at Ser(43) and Ser(510). Furthermore, introduction of S301A,S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E,S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.

FIGURE 1.

Identification of a region in Runx2 necessary for ERK/MAPK-dependent transcriptional activation and phosphorylation. A, schematic of the domain structure of Runx2 with relevant serine residues indicated. AD1–3, transcriptional activation domains; QA, glutamine/alanine-rich domain; RUNT, runt/DNA-binding domain; NLS, nuclear localization sequence; P/S/T domain, proline/serine/threonine-rich domain; RD, repressor domain (from Ref. 30). B, MAPK-dependent transcriptional activity. COS7 cells were transfected with wild type (WT) Runx2 or the indicated C-terminal deletions in the presence of control (LacZ) or Mek(sp) expression vectors and a 6OSE2-luc reporter as described under “Experimental Procedures.” Firefly luciferase activity was normalized for transfection efficiency using a R. reinformis luciferase plasmid. Asterisk, significantly different from corresponding control, p < 0.01; brackets indicate comparisons made; error bars, ±S.D. C and D, Runx2 phosphorylation. COS7 cell cultures treated as in B were metabolically labeled with [³²P]orthophosphate or Tran³⁵S-label as described under “Experimental Procedures.” Runx2 was immunoprecipitated (C) and ³²P incorporation was normalized to total ³⁵S-labeled protein in each group and expressed as fold-increase with Mek(sp) stimulation (D).

FIGURE 2.

Runx2 phosphorylation sites. A, in vitro peptide phosphorylation. Synthetic peptides were prepared containing amino acid residues 264–337 of the Runx2 sequence (indicated by arrows) or the indicated amino acid substitutions (top panel). Peptides were labeled with [γ-³²P]ATP using activated ERK1 and resolved by SDS-PAGE as described under “Experimental Procedures” (lower panel). B, MS/MS analysis of ERK/MAPK-related phosphorylation sites. COS7 cells were transduced with adenoviruses encoding biotinylation-tagged Runx2, BirA biotin transferase, and Mek(sp) with (U0126 treatment) or without (in vivo phosphorylation) MAPK inhibitor. Runx2 as then purified as described under “Experimental Procedures” and subjected to MS/MS analysis. For in vitro phosphorylation, an aliquot of Runx2 purified from U0126-treated cells was incubated with the activated MAPK before MS/MS analysis. Phosphopeptides were identified using Mascot software. Correlation rate indicates the probability that the indicated peptide identification in the Runx2 sequence is correct. C, verification of Ser³¹⁹ phosphorylation. The peptic peptide LSQMTSPSIHSTTPLSSTRGRGL was selected to manually validate the phosphorylation event. Mascot search results indicated that acquired MS/MS spectra contain product ion data for both [M + 2H]²⁺ and [M + 3H]³⁺ charged states for this peptide. The full MS spectra of the [M + 3H]³⁺ charged state is shown in the left panel. The right panel shows the annotated MS/MS spectra of m/z = 819.05 (+3). Diagnostic Y₁₇⁺² and Y₁₈⁺² ions are indicated with m/z values of 856.8 and 940.2, respectively, which confirms the presence of a phosphate on Ser³¹⁹.

FIGURE 3.

Additional evidence for Runx2 phosphorylation at Ser³⁰¹. A, analysis of electrophoretic mobility of wild type (WT) truncated Runx2-(1–330) and Ser³⁰¹ and Ser³¹⁹ mutants. The indicated truncated Runx2 mutants were expressed in COS7 cells in the presence or absence of Mek(sp) and ERK1 expression vectors. Cell lysates were analyzed by SDS-PAGE with or without prior treatment with calf intestinal alkaline phosphatase (CIAP). Runx2 was detected by Western blotting. B, anti-P-serine antibody reactivity with full-length Runx2. FLAG-tagged WT Runx2 or the indicated mutants were expressed in COS7 cells in the presence (+) or absence (−) of Mek(sp) or in the presence of the MAPK inhibitor (+U0126). Nuclear extracts were then either immunoprecipitated with M2 antibody and probed with an anti-P-serine monoclonal antibody or immunoprecipitated (IP) with anti-P-serine and probed with M2.

FIGURE 4.

Association of Runx2 with ERK. A, co-immunoprecipitation of endogenous Runx2 and ERK. MC-4 cell nuclear extracts were immunoprecipitated with IgG, ERK, or Runx2 antibodies and blots were probed as indicated. B–D, identification of the ERK binding domain in Runx2. Wild type (WT) Runx2 or the indicated N-terminal (HA-tagged Runx2, B) or C-terminal Runx2 deletions (FLAG-tagged Runx2, C) were expressed in COS7 cells and immunoprecipitated (IP) with the indicated antibodies. Blots were then probed for Runx2 (HA or M2 antibodies) or total ERK. Panel C also shows immunoprecipitation results using either full-length or the 1–330 truncated Runx2 containing either wild type sequence (WT) or the S301A,S319A double mutation (SA). Panel D compares immunoprecipitation activity of WT Runx2 with an internal deletion containing a consensus ERK-binding D site (amino acid residues 201–215).

FIGURE 5.

Functional analysis of Runx2 phosphorylation sites. A, identification of phosphorylation sites necessary for MAPK responsiveness using Runx2-(1–330). Specific mutations were created in Runx2-(1–330) to generate S282A, S301A, or S319A mutants or the indicated combinations as well as an S301E,S319E mutant. Runx2 expression plasmids were transfected into COS7 cells in the presence or absence of Mek(sp) vector and luciferase reporters as described in the legend to Fig. 1. B, evaluation of requirement for Ser³⁰¹ and Ser³¹⁹ sites in the context of full-length Runx2 protein. S301A,S319A or S301E,S319E mutations were generated in full-length Runx2 and evaluated for Mek(sp) responsiveness as in panel A. C, FGF2-responsiveness of wild type (WT) and mutant Runx2. COS7 cells were transfected with wild type full-length Runx2 or the S301A,S319A mutant. After 24 h, cells were treated for an additional 24 h with FGF2 (50 ηg/ml) before luciferase activity was measured. Statistically significant differences are indicated: a, p < 0.05; b, p < 0.01. Error bars, ±S.D.

FIGURE 6.

Induction of osteoblast differentiation markers by wild type (WT) Runx2 and phosphorylation site mutants. An mTERT-immortalized cell line derived from Runx2^−/− calvaria was transfected with wild type and mutant Runx2 expression vectors and grown for 5 days in ascorbic acid-containing medium before measurement of Ocn (A) and Bsp (B) mRNAs by real-time reverse transcription-PCR. Indicated samples were also treated with U0126 12 h before harvest. The mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA in each sample. Runx2 protein in each group was measured on Western blots (WB) (C). S/A mut, S301A,S319A mutant; S/E mut, S301E,S319E mutant. Statistically significant differences are indicated: a, p < 0.05; b, p < 0.01. β-gal, β-galactosidase. Error bars, ±S.D.

FIGURE 7.

Induction of osteoblast differentiation by wild type Runx2 and the S301A,S319A phosphorylation site mutant. Adenovirus expression vectors containing wild type and S301A,S319A Runx2 mutants (SA) were used to transduce C3H10T1/2 mesenchymal cells. Cells were then grown in differentiation medium for the indicated times before measurement of alkaline phosphatase activity (ALP; A) or Ocn/Bsp mRNA levels (OCN and BSP; normalized to glyceraldehyde-3-phosphate dehydrogenase (GAP); C and D). Runx2 protein in each sample was measured on Western blots (D). Statistically significant differences are indicated: a, p < 0.05; b, p < 0.01. SA, S301A,S319A mutant. Error bars, ±S.D.