A GAPDH mutant defective in Src-dependent tyrosine phosphorylation impedes Rab2-mediated events.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple intracellular activities in addition to its role in gluconeogenesis. Indeed, we have reported that GAPDH is required for Rab2-mediated retrograde transport from vesicular tubular clusters (VTCs). These diverse GAPDH activities are the result of posttranslational modifications that confer a new function to the enzyme. In that regard, GAPDH is tyrosine phosphorylated by Src. To establish the functional significance of this modification for GAPDH activity in Rab2-dependent events, an amino acid substitution was made at tyrosine 41 (GAPDH Y41F). The inability of Src to phosphorylate purified recombinant GAPDH Y41F was confirmed in an in vitro kinase assay. The mutant was then employed in a quantitative membrane-binding assay that measures Rab2 recruitment of soluble components to VTCs. As we observed with GAPDH wild type, Rab2 promoted GAPDH Y41F binding to membranes in a dose-dependent manner, indicating that GAPDH tyrosine phosphorylation is not required for VTC association. However, GAPDH was tyrosine phosphorylated on VTCs. Importantly, GAPDH Y41F blocked vesicular stomatitis virus-G transport in an assay that reconstitutes endoplasmic reticulum to Golgi trafficking, indicating that phosphorylation of tyrosine 41 is essential for GAPDH activity in the early secretory pathway. The block in transport is because of the decreased binding of atypical protein kinase C iota/lambda to GAPDH Y41F, which reduces beta-coat protein association with the VTC and subsequent formation of Rab2-mediated retrograde vesicles. Our results suggest that Src plays a pivotal role in regulating the interaction of Rab2 effectors on the VTC.
Tisdale
Ellen J
EJ
Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, 6374 Scott Hall, Detroit, MI 48201, USA. etisdale@med.wayne.edu
Artalejo
Cristina R
CR
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*; D020693:rab2 GTP-Binding Protein / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D011994:Recombinant Proteins; D014443:Tyrosine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019061:src-Family Kinases; D020693:rab2 GTP-Binding Protein