Our long-term goal is to understand the mechanism of protein sorting and recycling from vesicular tubular clusters (VTCs). VTCs function as transport complexes delivering cargo from the ER to the Golgi complex. Moreover, these structures are the first site of segregation of the anterograde and retrograde pathways and thereby perform a critical function. The small GTPase Rab2 plays an essential role in this trafficking step by promoting formation of retrograde-directed vesicles that carry recycling cargo back to the ER. Rab2- mediated vesicle formation requires atypical PKCi/X and GAPDH. In this application we propose to characterize Rab2 mechanistically and to establish the role of Rab2 in events associated with VTCs.
The Specific Aims are: 1) to identify the protein components of the VTC subcompartment that binds Rab2, 2) to characterize the interaction between components of the Rab2 complex by mapping their reciprocal binding domains, and 3) elucidate the molecular mechanism of Rab2-dependent-retrograde vesicle budding from VTCs. The importance of investigating Rab2 function in secretion is underscored by the observation that elevated Rab2, PKC, and GAPDH expression correlates with high metastatic potential in various cancers. Since metastasis is a reflection of cytoskeletal reorganization and increased cell motility, it is noteworthy that Rab2 and its effectors influence microtubule dynamics. However, the precise function of Rab2, aPKCi/A, and GAPDH in the early secretory pathway and how these proteins contribute to the disease are unknown. The studies proposed in this application will develop a new and novel paradigm that couples Rab2-Apkc lA-GAPDH dysfunction with pathological consequences.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cell Development and Function Integrated Review Group (CDF)
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Shapiro, Bert I
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Wayne State University
Schools of Medicine
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Tisdale, Ellen J; Talati, Nikunj K; Artalejo, Cristina R et al. (2016) GAPDH binds Akt to facilitate cargo transport in the early secretory pathway. Exp Cell Res 349:310-319
Ikonomov, Ognian C; Sbrissa, Diego; Compton, Lauren M et al. (2015) The Protein Complex of Neurodegeneration-related Phosphoinositide Phosphatase Sac3 and ArPIKfyve Binds the Lewy Body-associated Synphilin-1, Preventing Its Aggregation. J Biol Chem 290:28515-29
Ikonomov, Ognian C; Sbrissa, Diego; Venkatareddy, Madhusudan et al. (2015) Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis. Biochim Biophys Acta 1853:1240-50
Tisdale, Ellen J; Shisheva, Assia; Artalejo, Cristina R (2014) Overexpression of atypical protein kinase C in HeLa cells facilitates macropinocytosis via Src activation. Cell Signal 26:1235-42
Tisdale, Ellen J; Azizi, Fouad; Artalejo, Cristina R (2009) Rab2 utilizes glyceraldehyde-3-phosphate dehydrogenase and protein kinase C{iota} to associate with microtubules and to recruit dynein. J Biol Chem 284:5876-84
Tisdale, Ellen J; Artalejo, Cristina R (2006) 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. J Biol Chem 281:8436-42
Tisdale, Ellen J (2005) Rab2 purification and interaction with protein kinase C iota/lambda and glyceraldehyde-3-phosphate dehydrogenase. Methods Enzymol 403:381-91