Abstract
Microtubules display dynamic turnover during cell migration, leading to cell contractility and focal adhesion maturation regulated by Rho guanosine triphosphatase activity. This interplay between microtubules and actomyosin is mediated by guanine nucleotide exchange factor (GEF)-H1 released after microtubule depolymerization or microtubule disconnection from focal adhesions. However, how GEF-H1 activates Rho upon microtubule disassembly remains elusive. Here, we found that BNIP-2, a BCH domain-containing protein that binds both RhoA and GEF-H1 and traffics with kinesin-1 on microtubules, is important for GEF-H1-driven RhoA activation upon microtubule disassembly. Depletion of BNIP-2 in MDA-MB-231 breast cancer cells decreases RhoA activity and promotes cell migration. Upon nocodazole-induced microtubule disassembly, the interaction between BNIP-2 and GEF-H1 increases, while knockdown of BNIP-2 reduces RhoA activation and cell rounding via uncoupling RhoA-GEF-H1 interaction. Together, these findings revealed that BNIP-2 couples microtubules and focal adhesions via scaffolding GEF-H1 and RhoA, fine-tuning RhoA activity and cell migration.
Highlights
Directional cell migration, an important step of cancer invasion and metastasis, requires dynamic changes of the cytoskeleton and cell- matrix adhesions, which are tightly regulated by Rho guanosine triphosphatases (GTPases; e.g., RhoA, Rac1, and Cdc42) [1]
A putative Rho-binding domain (RBD)–like region is mapped within BNIP-2 and Cdc42GAP homology (BCH) domain of BNIP-2, which is homologous to the RBD that was identified in BNIP-S by sequence analysis to various Rho-binding proteins
These results show that BNIP-2 interacts with RhoA via the RBD region in BCH domain
Summary
Directional cell migration, an important step of cancer invasion and metastasis, requires dynamic changes of the cytoskeleton and cell- matrix adhesions, which are tightly regulated by Rho guanosine triphosphatases (GTPases; e.g., RhoA, Rac, and Cdc42) [1]. Loss-of-function mutations in RhoA have been revealed in various cancers [6], and RhoA inactivation can promote colorectal cancer growth and skin tumor formation [7, 8]. Those findings suggest that RhoA can function as a tumor suppressor. Microtubule depolymerization results in actomyosin contractility through the activation of RhoA. GEF-H1 is a microtubule- binding RhoA-specific GEF that activates RhoA when it is released from microtubules [10, 11] It plays a critical role in focal adhesion dynamics and mechanosensing for migrating cells [12]. It is known that GEF-H1 is spatially inhibited by microtubules [14] and microtubule capture by adhesion through KANK [15], less is known about how GEF-H1 activates RhoA after being released from microtubules and whether any scaffold proteins are involved in the process
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