Phosphorylation of Stim1 at serine 575 via netrin-2/Cdo-activated ERK1/2 is critical for the promyogenic function of Stim1.
The promyogenic cell surface molecule Cdo is required for activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells c3 (NFATc3) induced by netrin-2 in myogenic differentiation. However, the molecular mechanism leading to NFATc3 activation is unknown. Stromal interaction molecule 1 (Stim1), an internal calcium sensor of the endoplasmic reticulum store, promotes myogenesis via activation of NFATc3. In this study we investigated the functional interaction between Cdo and Stim1 in myogenic differentiation. Overexpression and depletion of Stim1 enhanced or decreased myotube formation, respectively. Of interest, Stim1 protein levels were decreased in Cdo-deficient perinatal hindlimb muscles or primary myoblasts; this correlates with defective NFATc3 activation in Cdo(-/-) myoblasts upon differentiation. Forced activation of NFATc3 by overexpression of calcineurin restored differentiation of Cdo-depleted C2C12 myoblasts. Furthermore, Cdo and Stim1 formed a complex in 293T cells or in differentiating C2C12 myoblasts. The netrin-2-mediated NFATc3 activation was coincident with robust interactions between Cdo and Stim1 in myoblasts and the ERK-mediated Stim1 phosphorylation at serine 575. The serine 575 phosphorylation was enhanced in C2C12 cells upon differentiation, and the alanine substitution of serine 575 failed to restore differentiation of Stim1-depleted myoblasts. Taken together, the results indicate that cell adhesion signaling triggered by netrin-2/Cdo induces Stim1 phosphorylation at serine 575 by ERK, which promotes myoblast differentiation.
Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.
Hye-Jin
HJ
Lee
Gyu-Un
GU
Bae
Young-Eun
YE
Leem
Hyun-Kyung
HK
Choi
Tong Mook
TM
Kang
Hana
H
Cho
Seong-Tae
ST
Kim
Jong-Sun
JS
Kang
D000818:Animals; D001483:Base Sequence; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D015220:Calcium Channels; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D024510:Muscle Development / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D050778:NFATC Transcription Factors / Q000378:metabolism; D009414:Nerve Growth Factors / Q000378:metabolism*; D000075385:Netrins; D010766:Phosphorylation; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000737:chemistry; D000071737:Stromal Interaction Molecule 1
D015220:Calcium Channels; C510744:Cdon protein, mouse; D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D046912:Multiprotein Complexes; D050778:NFATC Transcription Factors; D009414:Nerve Growth Factors; D000075385:Netrins; C494009:Nfatc3 protein, mouse; D034741:RNA, Small Interfering; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; C088894:netrin-2; D012694:Serine; D019703:Calcineurin