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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02606

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Selective cellular effects of overexpressed pleckstrin-homology domains that recognize PtdIns(3,4,5)P₃ suggest their interaction with protein binding partners

¹ Endocrinology and Reproduction Research Branch, National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
² Department of Physiology, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary
³ Department of Medical Chemistry, Faculty of Medicine, Semmelweis University, 1085 Budapest, Hungary

^* Author for correspondence (e-mail: ballat{at}mail.nih.gov)

Accepted 2 August 2005

Several pleckstrin-homology (PH) domains with the ability to bind phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P₃, PIP₃] were expressed as green fluorescent protein (GFP) fusion proteins to determine their effects on various cellular responses known to be activated by PIP₃. These proteins comprised the PH domains of Akt, ARNO, Btk or GRP1, and were found to show growth-factor-stimulated and wortmannin-sensitive translocation from the cytosol to the plasma membrane in several cell types, indicating their ability to recognize PIP₃. Remarkably, although overexpressed Akt-PH–GFP and Btk-PH–GFP were quite potent in antagonizing the PIP₃-mediated activation of the Akt protein kinase, such inhibition was not observed with the other PH domains. By contrast, expression of the PH domains of GRP1 and ARNO, but not of Akt or Btk, inhibited the attachment and spreading of freshly seeded cells to culture dishes. Activation of PLC by epidermal growth factor (EGF) was attenuated by the PH domains of GRP1, ARNO and Akt, but was significantly enhanced by the Btk PH domain. By following the kinetics of expression of the various GFP-fused PH domains for several days, only the PH domain of Akt showed a lipid-binding-dependent self-elimination, consistent with its interference with the anti-apoptotic Akt signaling pathway. Mutations of selective residues that do not directly participate in PIP₃ binding in the GRP1-PH and Akt-PH domain were able to reduce the dominant-negative effects of these constructs yet retain their lipid binding. These data suggest that interaction with and sequestration of PIP₃ may not be the sole mechanism by which PH domains interfere with cellular responses and that their interaction with other membrane components, most probably with proteins, allows a more specific participation in the regulation of specific signaling pathways.

Key words: PH domain, PI 3-kinase, PtdIns(3,4,5)P₃, PIP₃, Akt kinase, Cell adhesion, GFP

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