Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway.
The small GTPase Rab2 immunolocalizes to vesicular tubular clusters (VTCs) that function as transport complexes carrying cargo between the endoplasmic reticulum and the Golgi complex. Our previous studies showed that Rab2 promotes vesicle formation from VTCs and that the released vesicles are enriched in beta-coat protein, protein kinase C iota/lambda (PKCiota/lambda), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCiota/lambda kinase activity was necessary for vesicle formation, a search was initiated to identify the substrate(s) that potentiate Rab2 function within VTCs. In this study, we found that PKCiota/lambda phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCiota/lambda regulatory domain. Based on numerous observations that show (beta-COP) GAPDH associates with cytoskeletal elements, we examined the role of phospho-GAPDH in promoting microtubule (MT) binding to membrane. Using a quantitative microsomal binding assay, we found that membrane association of beta-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCiota/lambda kinase activity. Furthermore, normal rat kidney cells transfected with a constitutively activated form of Rab2 (Q65L) or with our anti-GAPDH polyclonal antibody displayed a dramatic change in MT organization. These combined results suggest that Rab2 stimulated PKCiota/lambda and GAPDH recruitment to VTCs, and the subsequent PKCiota/lambda phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.
Tisdale
Ellen J
EJ
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. etisdale@med.wayne.edu
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002846:Chromatography, Affinity; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007668:Kidney; D008861:Microsomes / Q000378:metabolism; D008870:Microtubules / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection; D020693:rab2 GTP-Binding Protein / Q000378:metabolism; D000099293:Protein Kinase C-lambda
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007527:Isoenzymes; D010446:Peptide Fragments; D011493:Protein Kinase C; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D020693:rab2 GTP-Binding Protein; D000099293:Protein Kinase C-lambda