Abstract
We previously identified a novel Rab small GTPase protein, Rab37, which plays a critical role in regulating exocytosis of secreted glycoproteins, tissue inhibitor of metalloproteinases 1 (TIMP1) to suppress lung cancer metastasis. Patients with preserved Rab37 protein expression were associated with better prognosis. However, a significant number of the patients with preserved Rab37 expression showed poor survival. In addition, the molecular mechanism for the regulation of Rab37-mediated exocytosis remained to be further identified. Therefore, we investigated the molecular mechanism underlying the dysregulation of Rab37-mediated exocytosis and metastasis suppression. Here, we report a novel mechanism for Rab37 inactivation by phosphorylation. Lung cancer patients with preserved Rab37, low TIMP1, and high PKCα expression profile correlate with worse progression-free survival examined by Kaplan-Meier survival, suggesting that PKCα overexpression leads to dysfunction of Rab37. This PKCα-Rab37-TIMP1 expression profile predicts the poor outcome by multivariate Cox regression analysis. We also show that Rab37 is phosphorylated by protein kinase Cα (PKCα) at threonine 172 (T172), leading to attenuation of its GTP-bound state, and impairment of the Rab37-mediated exocytosis of TIMP1, and thus reduces its suppression activity on lung cancer cell motility. We further demonstrate that PKCα reduces vesicle colocalization of Rab37 and TIMP1, and therefore inhibits Rab37-mediated TIMP1 trafficking. Moreover, Phospho-mimetic aspartate substitution mutant T172D of Rab37 significantly promotes tumor metastasis in vivo. Our findings reveal a novel regulation of Rab37 activity by PKCα-mediated phosphorylation which inhibits exocytic transport of TIMP1 and thereby enhances lung tumor metastasis.
Highlights
Rab small GTPases consist of the largest subgroup of Ras superfamily and function as regulators of intracellular vesicle transport, such as endocytosis, receptor recycling and exocytosis [1,2,3]
We previously reported that preserved expressions of metastatic suppressor Rab37 and its cargo, tissue inhibitor of metalloproteinases 1 (TIMP1), were associated with better prognosis [18]
Patients with preserved Rab37 expression and low TIMP1 expression were significantly associated with protein kinase Cα (PKCα) overexpression, supporting our hypothesis that PKCα overexpression leads to dysfunction of Rab37
Summary
Rab small GTPases consist of the largest subgroup of Ras superfamily and function as regulators of intracellular vesicle transport, such as endocytosis, receptor recycling and exocytosis [1,2,3]. We have previously cloned human Rab and found that Rab mRNA was down-regulated by promoter hypermethylation in non-small cell lung cancer patients. We further demonstrate that expression of Rab suppresses tumor progression by mediating exocytosis of TIMP1 and TSP1 to inactivate extracellular metalloproteinase 9 and to inhibit tumor neovasculature respectively [18, 19]. We show that Rab phosphorylation by PKCα inactivates Rab37-mediated exocytic transport by decreasing GTP-bound active form and thereby enhances lung tumor invasion and metastasis
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