Grp94 is Tyr-phosphorylated by Fyn in the lumen of the endoplasmic reticulum and translocates to Golgi in differentiating myoblasts.
The endoplasmic-reticulum chaperone Grp94 is required for the cell surface export of molecules involved in the native immune response, in mesoderm induction and muscle development, but the signals responsible for Grp94 recruitment are still obscure. Here we show for the first time that Grp94 undergoes Tyr-phosphorylation in differentiating myogenic C2C12 cells. By means of phospho-proteomic and immunoprecipitation analyses, and the use of Src-specific inhibitors we demonstrate that the Src-tyrosine-kinase Fyn becomes active early after induction of C2C12 cell differentiation, in parallel with the recruitment and the Tyr-phosphorylation of Grp94, which peaks at 6-hour differentiation. Grp94 is Tyr-phosphorylated inside the endoplasmic reticulum by a lumenal Fyn, as indicated by fluorescence and electronmicroscopy immunolocalization, co-immunoprecipitation after chemical cross-linking and by treatment of intact endoplasmic-reticulum vesicles with proteinase K. Furthermore, fractionation of cellular membrane compartments and double-immunofluorescence studies showed that Tyr-phosphorylation of Grp94 is necessary for the protein translocation from the endoplasmic reticulum to the Golgi apparatus. These results indicate that Fyn-catalyzed Tyr-phosphorylation of Grp94 is an event required to promote the chaperone export from the endoplasmic reticulum occurring in the early phase of myoblast differentiation.
Frasson
Martina
M
Department of Biochemistry, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy.
Vitadello
Maurizio
M
Brunati
Anna Maria
AM
La Rocca
Nicoletta
N
Tibaldi
Elena
E
Pinna
Lorenzo A
LA
Gorza
Luisa
L
Donella-Deana
Arianna
A
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D032446:Myoblasts / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000378:metabolism
D008562:Membrane Glycoproteins; C053387:endoplasmin; D019000:Phosphotyrosine; C494960:Fyn protein, mouse; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases