F-BAR Domain Protein PACSIN2

PACSIN2 and Rac1

The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins. Scientist identified a series of regulatory proteins that associate with Rac1 through its hypervariable C-terminal domain, including the Rac1 activator β-Pix and the membrane adapter caveolin-1. Now a new study of the F-BAR domain protein PACSIN2 as a Rac1 interactor that regulates Rac1-mediated cell spreading and migration. Among the family of Rho GTPases, Rac1 is one of the most extensively studied members. Following activation, Rac1 can interact with a series of effector proteins to trigger downstream signaling. In Rac1 proteins, the C-terminal hypervariable region is important for subcellular targeting and the regulation of signaling output.

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PACSIN2 colocalizes with Rac1 on early endosomes. Given that PACSIN2 interacts with the small GTPase Rac1, it was important to establish whether and where PACSIN2 and Rac1 colocalize. Immunostaining of HeLa cells for endogenous PACSIN2 and endogenous Rac1 showed that both proteins are present at perinuclear vesicles, and identified them as early endosomes. In addition to the colocalization of endogenous Rac1 and PACSIN2 on early endosomes, the endogenous Rac1 was localized to PACSIN2 tubules. Furthermore, in live cell imaging experiments, both proteins were found to coexist on endosomal structures in cells transduced with mCherry-rac1WT and YFP-PACSIN2.

PACSIN2 is a negative regulator of Rac1 signaling. PACSIN2 is an important regulator of the small GTPase Rac1 and that PACSIN2 limits Rac1-GTP signaling by promoting Rac1 inactivation. This effect of PACSIN2 requires its Rac1-binding capacity, mediated by the SH3 domain, as well as its membrane-tubulating activity, residing in the F-BAR domain. PACSIN2 is involved in the inactivation of Rac1 in a fashion that requires an intact FBAR domain. As the F-BAR domain does not regulate the PACSIN2-Rac1 interaction, this suggests that the membrane binding and/or tubulating activity of PACSIN2 is essential to inactivate Rac1. In addition, study showed that inhibition of dynamin impairs PACSIN2 function and localization and prevents PACSIN2-mediated Rac1 inactivation. Together, these data suggest a model in which PACSIN2 regulates Rac1 by promoting its internalization. In other studies, dynamin bound to PACSIN2 was previously shown to regulate Rac1, and further studies found that dominant-negative dynamin K44 mutants increase the formation of Rac1-positive tubule structures. Given that dynamin induces tubule fission and generates intracellular vesicles, such as early endosomes, dynamin may act downstream of PACSIN2 in the Rac1-inactivation pathway.

Fig 1. Rac1 signaling regulates PACSIN2 distribution.
(Source: Journal of Cell Science, March 2011.)

Subcellular distribution of PACSIN2 correlates with Rac1 activity. Activation of endogenous Rac1 with cytotoxic necrotizing factor 1(CNF1) toxin or expression of a constitutively active mutant of Rac1 (Q61L) results in loss of PACSIN2-positive tubular structures, leaving PACSIN2 on early endosomes, and activated Rac1 reduces the number of PACSIN2-positive tubules, which could reflect a reduction in PACSIN2-mediated endocytosis, whereas Rac1 inhibition led to an increase in the number of PACSIN2-mediated endocytosis, suggesting that Rac1 activity regulates the localization of PACSIN2 and the abundance of PACSIN2 positive tubular structures.