Biomedical subjects

Ellen J Tisdale

Publications and source records attributed to Ellen J Tisdale.

7 recordsLinked to original sources

PubMed2006

Src-dependent aprotein kinase C iota/lambda (aPKCiota/lambda) tyrosine phosphorylation is required for aPKCiota/lambda association with Rab2 and glyceraldehyde-3-phosphate dehydrogenase on pre-golgi intermediates.

The small GTPase Rab2 is required for membrane transport between the endoplasmic reticulum (ER) and the Golgi complex. Rab2 associates with pre-Golgi intermediates (also termed vesicular tubular clusters; VTCs) that sort cargo to the anterograde pathway from recycling proteins retrieved to the ER. Our previous studies have shown that Rab2 stimulates atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) recruitment to VTCs. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and aPKCiota/lambda and GAPDH interact. Based on the reports demonstrating aPKCiota-Src interaction and Src activity in the retrograde pathway (Golgi-ER), studies were initiated to learn whether Rab2 also promoted Src recruitment to VTCs. Using a quantitative membrane binding assay, we found that Rab2-stimulated Src membrane association in a dose-dependent manner. The recruited Src binds to aPKCiota/lambda and GAPDH on the membrane; however, Src does not interact with Rab2. The membrane-associated Src tyrosine phosphorylates aPKCiota/lambda on the VTC. To determine the consequence of aPKCiota/lambda tyrosine phosphorylation, the membrane binding assay was supplemented with the Src-specific tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2). Although Rab2, Src, and GAPDH recruitment was not affected, the Rab2-PP2-treated membranes contained a negligible amount of aPKCiota/lambda. Since Rab2 requires aPKCiota/lambda for the downstream recruitment of beta-coat protein (beta-COP) to VTCs, the Rab2-PP2-treated membranes were evaluated for the presence of beta-COP. Like aPKCiota/lambda, the membranes contained a negligible amount of beta-COP that was reflected by the drastic reduction in Rab2-dependent vesicle formation. These data suggest that Src-mediated tyrosine phosphorylation of aPKCiota/lambda facilitates aPKCiota/lambda association with Rab2-Src-GAPDH on VTCs, which is ultimately necessary for the downstream recruitment of beta-COP and release of Rab2-mediated retrograde-directed vesicles.

Animals↗

PubMed2005

Rab2 purification and interaction with protein kinase C iota/lambda and glyceraldehyde-3-phosphate dehydrogenase.

The small GTPase Rab2 is essential for membrane trafficking in the early secretory pathway. Rab2 associates with vesicular tubular clusters (VTCs) located between the endoplasmic reticulum (ER) and the Golgi complex. VTCs function as transport intermediates and sort anterograde-directed cargo from recycling proteins. Rab2 selectively recruits atypical protein kinase C iota/lambda (aPKCiota/lambda) and glyceraldehyde-3-phosphate (GAPDH) to VTCs where aPKCiota/lambda phosphorylates GAPDH. Both aPKCiota/lambda and GAPDH bind directly to Rab2 and this interaction ultimately results in COPI recruitment and the release of retrograde-directed vesicles. This chapter describes a protocol to purify recombinant Rab2 from Rab2 cDNA transformed bacteria and methods to assess recombinant Rab2 biological activity. Additionally, in vivo and in vitro assays are outlined that are employed to demonstrate Rab2 interaction with the downstream effectors aPKCiota/lambda and GAPDH.

Animals↗

PubMed2004

Glyceraldehyde-3-phosphate dehydrogenase interacts with Rab2 and plays an essential role in endoplasmic reticulum to Golgi transport exclusive of its glycolytic activity.

Rab2 requires atypical protein kinase C iota/lambda (aPKC iota/lambda) to promote vesicle formation from vesicular tubular clusters (VTCs). The Rab2-generated vesicles are enriched in recycling proteins suggesting that the carriers are retrograde-directed and retrieve transport machinery back to the endoplasmic reticulum. These vesicles also contained the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We have previously established that GAPDH is required for membrane transport between the endoplasmic reticulum and the Golgi complex. Moreover, GAPDH is phosphorylated by aPKC iota/lambda and binds to the aPKC iota/lambda regulatory domain. In this study, we employed a combination of in vivo and in vitro assays and determined that GAPDH also interacts with Rab2. The site of GAPDH interaction was mapped to Rab2 residues 20-50. In addition to its glycolytic function, GAPDH has multiple intracellular roles. However, the function of GAPDH in the early secretory pathway is unknown. One possibility is that GAPDH ultimately provides energy in the form of ATP. To determine whether GAPDH catalytic activity was critical for transport in the early secretory pathway, a conservative substitution was made at Cys-149 located at the active site, and the mutant was biochemically characterized in a battery of assays. Although GAPDH (C149G) has no catalytic activity, Rab2 recruited the mutant protein to membranes in a quantitative binding assay. GAPDH (C149G) is phosphorylated by aPKC iota/lambda and binds directly to Rab2 when evaluated in an overlay binding assay. Importantly, VSV-G transport between the ER and Golgi complex is restored when an in vitro trafficking assay is performed with GAPDH-depleted cytosol and GAPDH (C149G). These data suggest that GAPDH imparts a unique function necessary for membrane trafficking from VTCs that does not require GAPDH glycolytic activity.

Amino Acid Sequence↗

PubMed2003

Rab2 interacts directly with atypical protein kinase C (aPKC) iota/lambda and inhibits aPKCiota/lambda-dependent glyceraldehyde-3-phosphate dehydrogenase phosphorylation.

Atypical protein kinase C iota/lambda (PKCiota/lambda) is essential for protein transport in the early secretory pathway. The small GTPase Rab2 selectively recruits the kinase to vesicular tubular clusters (VTCs) where PKCiota/lambda phosphorylates glyceraldehyde-3-phosphate dehydrogenase (GAPDH). VTCs are composed of small vesicles and tubules and serve as transport intermediates that shuttle cargo from the endoplasmic reticulum to the Golgi complex. These structures are the first site of segregation of the anterograde and retrograde pathways. When Rab2 binds to a VTC subcompartment, the subsequent recruitment of PKCiota/lambda and soluble components, including COPI (coatomer and ADP-ribosylation factor), results in the release of retrograde-directed vesicles. Because Rab2 stimulates PKCiota/lambda membrane association in a dose-dependent manner, we investigated whether the two proteins physically interact. Using a combination of in vivo and in vitro assays, we found that Rab2 interacts directly with PKCiota/lambda and that this interaction occurs through the Rab2 amino terminus (residues 1-19) and the PKCiota/lambda regulatory domain. A mutant lacking the PKCiota/lambda binding domain (Rab2N'Delta19) was functionally characterized. In contrast to Rab2, Rab2N'Delta19 failed to recruit PKCiota/lambda to normal rat kidney microsomes in a quantitative binding assay. To determine whether Rab2 modulates the ability of PKCiota/lambda to phosphorylate GAPDH, an in vitro kinase assay was supplemented with Rab2 or Rab2N'Delta19. Rab2 inhibited PKCiota/lambda-dependent GAPDH phosphorylation, whereas no effect was observed when the assay was performed with the aminoterminal truncation mutant. These results suggest that a downstream effector recruited to the VTC stimulates PKCiota/lambda-mediated GAPDH phosphorylation by alleviating the inhibition imposed by Rab2-PKCiota/lambda interaction.

Animals↗

PubMed2003

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates.

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.

Amino Acid Sequence↗

PubMed2003

The N'-terminal domain of glyceraldehyde-3-phosphate dehydrogenase of the apicomplexan Plasmodium falciparum mediates GTPase Rab2-dependent recruitment to membranes.

Spatial and temporal distribution of the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (pfGAPDH) and aldolase (pfAldolase) of Plasmodium falciparum were investigated using specific mAbs and indirect immunofluorescence analysis (IFA). Both glycolytic enzymes were co-localized during ring and trophozoite stages of both liver and asexual blood stage parasites. During schizogony, pfGAPDH became associated with the periphery of the parasites and eventually accumulated in the apical region of merozoites, while pfAldolase showed no segregation. Subcellular fractionation experiments demonstrated that pfGAPDH was found in both the membrane-containing pellet and the supernatant fraction of parasite lysates. In contrast, pfAldolase was only found in the supernatant fraction. A quantitative binding assay showed that pfGAPDH could be recruited to HeLa cell microsomal membranes in response to mammalian GTPase Rab2, indicating that Rab2-dependent recruitment of cytosolic components to membranes is conserved in evolution. Two overlapping fragments of pfGAPDH (residues 1-192 and 133-337) were evaluated in the microsomal binding assay. We found that the N'-terminal fragment competitively inhibited Rab2-stimulated pfGAPDH recruitment. Thus, the domain mediating the evolutionarily conserved Rab2-dependent membrane recruitment is located in the N'-terminus of GAPDH. Together, these results suggest that pfGAPDH exerts non-glycolytic function(s) in P. falciparum, possibly including a role in vesicular transport and biogenesis of apical organelles.

Animals↗

PubMed2001

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.

Amino Acid Sequence↗