The RLIP76 N-terminus binds ARNO to regulate PI 3-kinase, Arf6 and Rac signaling, cell spreading and migration

Biochem Biophys Res Commun. 2014 Nov 28;454(4):560-5. doi: 10.1016/j.bbrc.2014.10.114. Epub 2014 Oct 30.

Abstract

RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.

Keywords: ARF; Cell migration; Cell signaling; PI 3-kinase (PI3K); Rac.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • ADP-Ribosylation Factors / metabolism*
  • Animals
  • Binding Sites
  • Cell Movement*
  • Cells, Cultured
  • GTPase-Activating Proteins / metabolism*
  • Mice
  • NIH 3T3 Cells
  • Phosphatidylinositol 3-Kinases / metabolism*
  • Signal Transduction*
  • rac GTP-Binding Proteins / metabolism*

Substances

  • GTPase-Activating Proteins
  • Ralbp1 protein, mouse
  • cytohesin-2
  • Phosphatidylinositol 3-Kinases
  • ADP-Ribosylation Factors
  • ADP-ribosylation factor 6
  • rac GTP-Binding Proteins