Abstract
The intracellular signaling pathways through ADP-ribosylation factors (Arfs) of the small GTPase family control cell morphological changes by regulating membrane components and/or cytoskeletal protein dynamics. We previously reported that cytohesin-2 (CYTH2), an Arf-guanine-nucleotide exchange factor (GEF), binds to the cytoskeletal scaffold protein paxillin through C-terminal region of CYTH2 and promotes the migration of mouse 3T3-L1 fibroblasts. In mammals, CYTH family GEFs are composed of four subfamilies. Among them, CYTH2 and CYTH3 are widely expressed in tissues and it remains to be clarified to determine whether they have specific biochemical and cellular functions or are redundant. Here, we show that the C-terminal short polybasic region of CYTH2 is necessary and sufficient for binding to paxillin to mediate cell migration. Although 3T3-L1 cells primarily express CYTH2 and CYTH3 of four CYTH family members, neither knockdown of CYTH3 by the specific siRNA nor expression of its C-terminal region inhibits migration. Importantly, replacing the C-terminal region of CYTH3 with that of CYTH2 adds the ability of paxillinbinding and mediating migration to CYTH3. Conversely, replacing the C- terminal region of CYTH2 with that of CYTH3 leads to loss of these abilities of CYTH2. These results reveal that paxillin is a unique binding partner with CYTH2 in migrating cells, presenting the first CYTH family GEF’s region that is involved in the selectivity of the binding protein.
Highlights
Cell migration is a crucial process for early development and is seen in pathological states
We previously reported that CYTH2, through its C-terminal region containing the PH domain and the C-terminal polybasic region, binds to the cytoskeletal scaffold protein paxillin and mediates the migration of mouse 3T3L1 fibroblasts [11]
This conclusion is supported by the result that replacing the C-terminal region of CYTH3 with that of CYTH2 adds the ability to bind to paxillin to mediate the migration to CYTH3 though it does not work the other way around
Summary
Cell migration is a crucial process for early development and is seen in pathological states. It is established that the intracellular signaling mechanism responsible for cell morphological changes, such as migration and morphological differentiation, are controlled by small GTPases of the Rho family [1,2,3,4]. Recent evidence demonstrates that in addition to Rho GTPases, Arf branches of small GTPases are involved in cell morphological changes [5,6,7,8]. Arf and Arf are well characterized to interact with the downstream effectors, including lipid-modifying enzymes such as phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks) and phospholipase D isozymes (PLDs), and coat proteins, which coordinately contribute to cell morphological changes through modification of membrane lipid components and/or modulation of actin cytoskeletal dynamics [5,6,7,8]
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