Abstract
3-Phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates the T-loop of several AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family protein kinases, resulting in their activation. Previous structural studies have revealed that the alpha C-helix, located in the small lobe of the kinase domain of PDK1, is a key regulatory element, as it links a substrate interacting site termed the hydrophobic motif (HM) pocket with the phosphorylated Ser-241 in the T-loop. In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the alpha C-helix are not required for PDK1 activity or substrate binding through the HM-pocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site. The structure of an inactive T-loop mutant of PDK1, in which Ser-241 is changed to Ala, was also determined. This structure, together with surface plasmon resonance binding studies, demonstrates that the PDK1(S241A)-inactive mutant possesses an intact HM-pocket as well as an ordered alpha C-helix. These findings reveal that the integrity of the alpha C-helix and HM-pocket in PDK1 is not regulated by T-loop phosphorylation.
Highlights
3-Phosphoinositide-dependent protein kinase-1 (PDK1)1 is a Ser/Thr protein kinase that activates at least 23 protein kinases belonging to the AGC family of protein kinases by phosphoryl
In this study we have demonstrated by mutational analysis that interactions between the phosphorylated Ser-241 and the ␣C-helix are not required for PDK1 activity or substrate binding through the HMpocket but are necessary for PDK1 to be activated or stabilized by a peptide that binds to this site
We have investigated how PDK1 T-loop phosphorylation affects activation and peptide binding in the hydrophobic motif (HM)-pocket
Summary
3-Phosphoinositide-dependent protein kinase-1 (PDK1) is a Ser/Thr protein kinase that activates at least 23 protein kinases belonging to the AGC (cAMP-dependent, cGMP-dependent, protein kinase C) family of protein kinases by phosphoryl-. The other PDK1 AGC kinase substrates, including p70 ribosomal S6-kinase (8), serum- and glucocorticoid-responsive kinase (9), and p90 ribosomal S6 kinase (10), do not bind PtdIns(3,4,5)P3/ PtdIns(3,4)P2 or possess PH domains Instead, these kinases possess conserved PDK1 docking sites located in a C-terminal region known as the hydrophobic motif (HM). The structural analysis of the PDK1 kinase domain has revealed that, similar to what has been observed in other kinases, the phosphorylated Ser-241 residue forms key interactions coordinating and aligning important catalytic motifs such as the ␣C-helix of the N-terminal lobe. In PDK1 it positions the conserved Glu-130 residue such that it coordinates the conserved Lys-111, which interacts with the ␣-phosphate of ATP This hydrogen bonding network is conserved in most protein kinases and is required for phosphoryl transfer. PDK1-mediated phosphorylation of PKB and interaction of PKB with its own hydrophobic motif (HM) led to
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