The in vivo role of PtdIns(3,4,5)P3 binding to PDK1 PH domain defined by knockin mutation

Abstract

We generated homozygous knockin ES cells expressing a form of 3-phosphoinositide-dependent protein kinase-1 (PDK1) with a mutation in its pleckstrin homology (PH) domain that abolishes phosphatidylinositol 3,4,5-tris-phosphate (PtdIns(3,4,5)P3) binding, without affecting catalytic activity. In the knockin cells, protein kinase B (PKB) was not activated by IGF1, whereas ribosomal S6 kinase (RSK) was activated normally, indicating that PtdIns(3,4,5)P3 binding to PDK1 is required for PKB but not RSK activation. Interestingly, amino acids and Rheb, but not IGF1, activated S6K in the knockin cells, supporting the idea that PtdIns(3,4,5)P3 stimulates S6K through PKB-mediated activation of Rheb. Employing PDK1 knockin cells in which either the PtdIns(3,4,5)P3 binding or substrate-docking 'PIF pocket' was disrupted, we established the roles that these domains play in regulating phosphorylation and stabilisation of protein kinase C isoforms. Moreover, mouse PDK1 knockin embryos in which either the PH domain or PIF pocket was disrupted died displaying differing phenotypes between E10.5 and E11.5. Although PDK1 plays roles in regulating cell size, cells derived from PH domain or PIF pocket knockin embryos were of normal size. These experiments establish the roles of the PDK1 regulatory domains and illustrate the power of knockin technology to probe the physiological function of protein-lipid and protein-protein interactions.

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…Phosphorylation of PKA T197 relative to total PKA was also slightly decreased in PDK1 −/− ES cells to PDK1 −/− +LG ES cells. Total levels of various PKC isoforms were also increased following expression of PDK1 L159G, consistent with previous reports [32]. We then analyzed phosphorylation of PDK1 substrates following incubation with the PP1 analogues 1-NM-PP1 and 3,4-DMB-PP1 in PDK1 −/− +LG cells.…”

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…In the same studies, the total levels of PKC were reported to be relatively stable in ES cells lacking PDK-1 and phosphorylation at the A-loop was abolished [37,38]. In T lymphocytes, Ghosh and colleagues have demonstrated that knockdown of PDK-1 expression inhibits A-loop phosphorylation of PKC [39]; this defect is recapitulated in PDK-1-null T cells [40].…”

“…PDK-1 has been shown to phosphorylate cPKC, nPKC and aPKCs both in vitro and when over-expressed in different cell types [33][34][35][36]. Most importantly perhaps is that the intracellular levels and/or A-loop phosphorylation of PKCs is [37,38].…”

“…While this phosphorylation site is not conserved in aPKCs, the negatively charged glutamic acid residue at the HM site on PKC performs a similar function [28]. The sequence surrounding the HM also serves as a docking site for the A-loop kinase PDK-1 [44,45,38].…”

The survival pathways phosphatidylinositol‐3 kinase (PI3‐K)/phosphoinositide‐dependent protein kinase 1 (PDK1)/Akt modulate liver regeneration through hepatocyte size rather than proliferation†

“…To confirm the role of PDK1-Akt/mTOR pathway in liver regeneration, we employed the "pif-pocket" mutant of PDK1, which allows PDK1 to signal exclusively to Akt but not to p70 S6K or others. 30,[34][35][36] Adenovirus-mediated introduction of the pif-pocket mutant (L155E) did re-phosphorylate Akt (Thr308), but not p70 S6K (Thr389), 4 hours after PH in L-Pdk1KO mice (Fig. 5A).…”