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

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 PLCγ 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.

Small molecule inhibition of phosphatidylinositol-3,4,5-triphosphate (PIP3) binding to pleckstrin homology domains

“…To begin characterizing cellular activities of PITs, we performed a PH domain translocation assay that measures PIP3-mediated association of the GFP-fused PH domains with the plasma membrane (5). As shown in Fig.…”

“…Membrane translocation and activation of the PIP3 target proteins initiate a variety of local responses, including assembly of signaling complexes and priming of protein kinase cascades (1,2). PIP3 regulates an array of PH domain-containing proteins (4), such as serine-threonine kinases Akt and PDK1, GRP1, a GDP/GTP exchange factor of ADP ribosylating factor 6, and protein tyrosine kinases of the Bruton's tyrosine kinase (Btk) and Tec families (5,6). This diversity in PIP3 signaling makes it one of the most important second messengers downstream from growth factor and oncogene signals.…”

Inhibition of PI3K induces Rac Activation and Membrane Ruffling in Proto-Dbl Expressing Cells

“…1B). Finally, coexpression of the PH domain of Akt (pCEFL-GST-Akt-PH), described previously to have a strong inhibitory effect on Akt activation and thus to function as a negative regulator of PI3K signaling, 19 also resulted in inhibition of focus formation (Fig. 1B).…”

Molecular and cellular bases of chronic myeloid leukemia

“…PTEN is a phosphatase that dephosphorylates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Maehama and Dixon, 1998;Stambolic et al, 1998). PIP3 is a direct product of PI3K activity, and plays a critical role in the regulation of cell survival and growth through activating the Ser/Thr protein kinase PDK1 and its downstream target Akt (Rameh and Cantley, 1999;Varnai et al, 2005). Loss of PTEN results in the development of AML and ALL in mice (Zhang et al, 2006).…”