Abstract
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 PLCgamma 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.
Highlights
Pleckstrin-homology (PH) domains are protein modules with a characteristic fold that have gained a great deal of interest as a result of their ability to bind phosphoinositides (Harlan et al, 1994; Lemmon et al, 1997; Lemmon, 2003)
Recognition of PIP3 by the PH domains in NIH 3T3 cells Four PH domains known to interact with PIP3 were chosen for this study: Btk-PH, Akt-PH, GRP1-PH and ARNO-PH
As documented by several studies, all of these proteins show a PIP3-dependent translocation to the plasma membrane in many cell types after agonist stimulation (Venkateswarlu et al, 1998; Oatey et al, 1999; Varnai et al, 1999; Watton and Downward, 1999)
Summary
Pleckstrin-homology (PH) domains are protein modules with a characteristic fold that have gained a great deal of interest as a result of their ability to bind phosphoinositides (Harlan et al, 1994; Lemmon et al, 1997; Lemmon, 2003). Several PH domains have been shown to be the main determinant of the recruitment of proteins to specific membrane compartments in a lipiddependent manner, leading to the idea that PH domains serve as localization signals responding to local formation of inositol phospholipids (Lemmon and Ferguson, 2000). Several observations suggest that PH domains might function in a more complex manner. In many proteins, such as in dynamin, the PH domain alone does not have high enough affinity to phosphoinositides to determine solely the localization of the protein, yet the protein shows clear regulation by inositol lipids (Artalejo et al, 1997; Szaszak et al, 2002; Yu et al, 2004). In spite of their great success in providing invaluable spatial and
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