Glyceraldehyde-3-phosphate Dehydrogenase Is Phosphorylated by Protein Kinase Cι/λ and Plays a Role in Microtubule Dynamics in the Early Secretory Pathway

Abstract 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 β-coat protein, protein kinase C ι/λ (PKCι/λ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the recycling protein p53/gp58. Because PKCι/λ 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 PKCι/λ phosphorylates GAPDH. Moreover, GAPDH interacts directly with the PKCι/λ regulatory domain. Based on numerous observations that show (β-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 β-tubulin was dependent on phospho-GAPDH and was blocked by reagents that interfere with Rab2-dependent GAPDH membrane recruitment or with PKCι/λ 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 PKCι/λ and GAPDH recruitment to VTCs, and the subsequent PKCι/λ phosphorylation of GAPDH ultimately influences MT dynamics in the early secretory pathway.