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Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.

ABSTRACT: Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 PH with substantial chemical shift changes that are much larger than PIP2. This suggests an enhanced association of kindlin-2 with membrane as mediated by PIP3 upon its conversion from PIP2 by phosphoinositide-3 kinase, a known regulator of integrin activation. We determined the NMR structure of the kindlin-2 PH domain bound to the head group of PIP3, inositol 1,3,4,5-tetraphosphate (IP4). The structure reveals a canonical PH domain fold, yet with a distinct IP4 binding pocket that appears highly conserved for the kindlin family members. Functional experiments demonstrate that although wild type kindlin-2 is capable of cooperating with integrin activator talin to induce synergistic integrin ?(IIb)?(3) activation, this ability is significantly impaired for a phosphoinositide binding-defective kindlin-2 mutant. These results define a specific PIP3 recognition mode for the kindlin PH domain. Moreover, they shed light upon a mechanism as to how the PH domain mediates membrane engagement of kindlin-2 to promote its binding to integrin and cooperation with talin for regulation of integrin activation.

SUBMITTER: Liu J

PROVIDER: S-EPMC3234820 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.

Liu Jianmin J Fukuda Koichi K Xu Zhen Z Ma Yan-Qing YQ Hirbawi Jamila J Mao Xian X Wu Chuanyue C Plow Edward F EF Qin Jun J

The Journal of biological chemistry 20111026 50

Kindlins are a subclass of FERM-containing proteins that have recently emerged as key regulators of integrin receptor activation and signaling. As compared with the conventional FERM domain, the kindlin FERM domain contains an inserted pleckstrin homology (PH) domain that recognizes membrane phosphoinositides, including phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). Using NMR spectroscopy, we show that PIP3 site-specifically binds to kindlin-2 P ...[more]

PMID: 22030399

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Project description:Numerous hormones, cytokines and transforming oncogenes activate phosphoinositide 3-kinase (PI-3K), a lipid kinase that initiates signal transduction cascades regulating cellular proliferation, survival, protein synthesis and glucose metabolism. PI-3K catalyses the production of the 3'-phosphoinositides PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3), which recruit downstream effector enzymes to the membrane via their pleckstrin homology (PH) domains. Recent studies have indicated that another signalling lipid, the sphingolipid ceramide, inhibits several PI-3K-dependent events, including insulin-stimulated glucose uptake and growth-factor-stimulated cell survival. Here we show that ceramide analogues specifically prevent the recruitment of the PtdIns(3,4,5)P(3)-binding proteins Akt/protein kinase B (PKB) or the general receptor for phosphoinositides-1 (GRP1). Specifically, the short-chain ceramide derivative C2-ceramide inhibited the platelet-derived growth factor (PDGF)-stimulated translocation of full-length Akt/PKB, as well as truncated proteins encoding only the PH domains of Akt/PKB or GRP1. C2-ceramide did not alter the membrane localization of the PH domain for phospholipase Cdelta, which preferentially binds PtdIns(4,5)P(2), nor did it affect the PDGF-stimulated production of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Interestingly, a glucosylceramide synthase inhibitor, 1-phenyl-2-decanoylamino-3-morpholinopropan-1-ol (PDMP), shown previously to increase intracellular ceramide concentrations without affecting PI-3K [Rani, Abe, Chang, Rosenzweig, Saltiel, Radin and Shayman (1995) J. Biol. Chem. 270, 2859-2867], recapitulated the inhibitory effects of C2-ceramide on PDGF-stimulated Akt/PKB phosphorylation. These studies indicate that ceramide prevents the translocation of certain PtdIns(3,4,5)P(3)-binding proteins, despite the presence of a full complement of PtdIns(3,4)P(2) or PtdIns(3,4,5)P(3). Furthermore, these findings suggest a mechanism by which stimuli that induce ceramide synthesis could negate the fundamental signalling pathways initiated by PI-3K.

Dataset Information

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.

Publications

Structural basis of phosphoinositide binding to kindlin-2 protein pleckstrin homology domain in regulating integrin activation.

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