Phosphorylation of Shox2 is required for its function to control sinoatrial node formation.
BACKGROUND
Inactivation of Shox2, a member of the short-stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown.


RESULTS
A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2-hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p-Shox2a) in cell culture as well as in the developing mouse and human SAN. Site-directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal-regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a-S92AS110A, mutant in the developing heart resulted in down-regulation of Nkx2.5 in wild-type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region.


CONCLUSIONS
Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.
Department of Cell and Molecular Biology, Tulane University, New Orleans, 70118, LA (H.L., C.H.C., W.Y., R.E.L., Y.P.C.).
Hongbing
H
Liu
Department of Cell and Molecular Biology, Tulane University, New Orleans, 70118, LA (H.L., C.H.C., W.Y., R.E.L., Y.P.C.).
Chao-Hui
CH
Chen
Department of Cell and Molecular Biology, Tulane University, New Orleans, 70118, LA (H.L., C.H.C., W.Y., R.E.L., Y.P.C.).
Wenduo
W
Ye
Department of Cell and Molecular Biology, Tulane University, New Orleans, 70118, LA (H.L., C.H.C., W.Y., R.E.L., Y.P.C.) Division of Cardiology, Children's Hospital Boston and Harvard Medical School, Boston, MA.
Ramón A
RA
Espinoza-Lewis
Center for Biomedical Research of South China, Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou, Fujian Province, China (X.H., Y.Z., Y.P.C.).
Xuefeng
X
Hu
Center for Biomedical Research of South China, Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou, Fujian Province, China (X.H., Y.Z., Y.P.C.).
Yanding
Y
Zhang
Department of Cell and Molecular Biology, Tulane University, New Orleans, 70118, LA (H.L., C.H.C., W.Y., R.E.L., Y.P.C.) Center for Biomedical Research of South China, Fujian Key Laboratory of Developmental and Neuro Biology, College of Life Science, Fujian Normal University, Fuzhou, Fujian Province, China (X.H., Y.Z., Y.P.C.).
YiPing
Y
Chen
D000818:Animals; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006339:Heart Rate / Q000502:physiology; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000502:physiology*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010766:Phosphorylation / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction; D012849:Sinoatrial Node / Q000196:embryology / Q000254:growth & development*; D014157:Transcription Factors / Q000502:physiology; D020798:Two-Hybrid System Techniques
D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; C095552:NKX2-5 protein, human; C484469:Nkx2-5 protein, mouse; C484664:SHOX2 protein, human; C484663:Shox2 protein, mouse; D014157:Transcription Factors
DNA binding; Nkx2.5 repression; SAN development; Shox2; pacemaking; phosphorylation