Tisdale Ellen J, Artalejo Cristina R
Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, 6374 Scott Hall, Detroit, MI 48201, USA.
Traffic. 2007 Jun;8(6):733-41. doi: 10.1111/j.1600-0854.2007.00569.x. Epub 2007 May 4.
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.
甘油醛-3-磷酸脱氢酶（GAPDH）除了在糖异生中发挥作用外，还具有多种细胞内活性。事实上，我们已经报道过，Rab2介导的从囊泡管状簇（VTCs）的逆行转运需要GAPDH。这些不同的GAPDH活性是翻译后修饰的结果，这些修饰赋予了该酶新的功能。在这方面，GAPDH被Src酪氨酸磷酸化。为了确定这种修饰对Rab2依赖性事件中GAPDH活性的功能意义，在酪氨酸41处进行了氨基酸替换（GAPDH Y41F）。在体外激酶试验中证实了Src无法磷酸化纯化的重组GAPDH Y41F。然后将该突变体用于定量膜结合试验，该试验测量Rab2将可溶性成分募集到VTCs的情况。正如我们在GAPDH野生型中观察到的那样，Rab2以剂量依赖性方式促进GAPDH Y41F与膜的结合，表明VTCs结合不需要GAPDH酪氨酸磷酸化。然而，GAPDH在VTCs上被酪氨酸磷酸化。重要的是，在重建内质网到高尔基体运输的试验中，GAPDH Y41F阻断了水疱性口炎病毒-G的运输，表明酪氨酸41的磷酸化对于早期分泌途径中GAPDH的活性至关重要。运输受阻是因为非典型蛋白激酶C iota/lambda与GAPDH Y41F的结合减少，这降低了β-包被蛋白与VTC的结合以及随后Rab2介导的逆行囊泡的形成。我们的结果表明，Src在调节Rab2效应器在VTC上的相互作用中起关键作用。
