Biochemical Journal

Accelerated Publication

Binding of phosphatidylinositol 3,4,5-trisphosphate to the pleckstrin homology domain of protein kinase B induces a conformational change

Christine C. MILBURN, Maria DEAK, Sharon M. KELLY, Nick C. PRICE, Dario R. ALESSI, Daan M. F. van AALTEN

Abstract

Protein kinase B (PKB/Akt) is a key regulator of cell growth, proliferation and metabolism. It possesses an N-terminal pleckstrin homology (PH) domain that interacts with equal affinity with the second messengers PtdIns(3,4,5)P3 and PtdIns(3,4)P2, generated through insulin and growth factor-mediated activation of phosphoinositide 3-kinase (PI3K). The binding of PKB to PtdIns(3,4,5)P3/PtdIns(3,4)P2 recruits PKB from the cytosol to the plasma membrane and is also thought to induce a conformational change that converts PKB into a substrate that can be activated by the phosphoinositide-dependent kinase 1 (PDK1). In this study we describe two high-resolution crystal structures of the PH domain of PKBα in a noncomplexed form and compare this to a new atomic resolution (0.98 Å, where 1 Å=0.1 nm) structure of the PH domain of PKBα complexed to Ins(1,3,4,5)P4, the head group of PtdIns(3,4,5)P3. Remarkably, in contrast to all other PH domains crystallized so far, our data suggest that binding of Ins(1,3,4,5)P4 to the PH domain of PKB, induces a large conformational change. This is characterized by marked changes in certain residues making up the phosphoinositide-binding site, formation of a short α-helix in variable loop 2, and a movement of variable loop 3 away from the lipid-binding site. Solution studies with CD also provided evidence of conformational changes taking place upon binding of Ins(1,3,4,5)P4 to the PH domain of PKB. Our data provides the first structural insight into the mechanism by which the interaction of PKB with PtdIns(3,4,5)P3/PtdIns(3,4)P2 induces conformational changes that could enable PKB to be activated by PDK1.

  • circular dichroism (CD)
  • crystallography
  • PDK1
  • phosphoinositide 3-kinase (PI3K)

Footnotes

  • Abbreviations used: diC8, dioctanoyl; GST, glutathione S-transferase; PDK1, phosphoinositide-dependent kinase 1; PEG, poly(ethylene glycol); PH, pleckstrin homology; PKB, protein kinase B; PI3K, phosphoinositide 3-kinase; RMSD, root-mean-square deviation.