Journal
JOURNAL OF CELL SCIENCE
Volume 120, Issue 5, Pages 792-801Publisher
COMPANY OF BIOLOGISTS LTD
DOI: 10.1242/jcs.03373
Keywords
centaurin-alpha(2); PI3-kinase; PIP3; PI 3,4-P-2; ARF6; actin; GAP
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Funding
- Biotechnology and Biological Sciences Research Council [BB/C515455/1] Funding Source: researchfish
- Medical Research Council [G0401232] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BB/C515455/1] Funding Source: Medline
- Medical Research Council [G0401232] Funding Source: Medline
- MRC [G0401232] Funding Source: UKRI
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GTPase activating proteins ( GAPs) of the centaurin family regulate the actin cytoskeleton and vesicle trafficking through inactivation of the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. We report the functional characterisation of centaurin-alpha(2), which is structurally related to the centaurin-alpha(1) ARF6 GAP. Centaurin-alpha(2) contains an N-terminal GAP domain followed by two pleckstrin homology (PH) domains (N-PH and C-PH). In vitro, GFP-centaurin-alpha(2) specifically binds the phosphatidylinositol (PI) 3-kinase lipid products, PI 3,4-P-2 and PI 3,4,5-P-3 (PIP3), through its C-terminal PH domain. In agreement with this observation, GFP-centaurin-alpha(2) 2 was recruited to the plasma membrane from the cytosol in EGF-stimulated cells in a PI-3-kinase-dependent manner. Moreover, the C-PH domain is sufficient and necessary for membrane recruitment of centaurin-alpha(2). Centaurin-alpha(2') shows sustained kinetics of PI-3-kinase-mediated membrane recruitment in EGF-stimulated cells, owing to its binding to PI 3,4-P-2. Centaurin-alpha(2) prevents ARF6 translocation to, and cortical actin formation at, the plasma membrane, which are phenotypic indications for ARF6 activation in EGF-stimulated cells. Moreover, the constitutively active mutant of ARF6 reverses the effect of centaurin-alpha(2) on cortical actin formation. The membrane targeted centaurin-alpha(2) is constitutively active. Together, these studies indicate that centaurin-alpha(2) is recruited in a sustained manner to the plasma membrane through binding to PI 3,4-P-2 and thereby regulates actin reorganisation via ARF6.
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