The PH Domain of Phosphoinositide-Dependent Kinase-1 Exhibits a Novel, Phospho-Regulated Monomer鈥揇imer Equilibrium with Important Implications for Kinase Domain Activation: Single-Molecule and Ensembl

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• 作者：Brian P. Ziemba ; Carissa Pilling ; V茅ronique Calleja ; Banafsh茅 Larijani ; Joseph J. Falke
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：July 16, 2013
• 年：2013
• 卷：52
• 期：28
• 页码：4820-4829
• 全文大小：458K
• 年卷期：v.52,no.28(July 16, 2013)
• ISSN：1520-4995

文摘

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer鈥搈onomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP₃ target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP₃-bound WT PH domain on membranes is predominantly dimeric while the PIP₃-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain鈥揚IP₃ complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP₃ binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows deeper insertion of the protein into the viscous bilayer, thereby increasing the diffusional friction. Ensemble measurements of PH domain affinity for PIP₃ on plasma membrane-like bilayers reveal that the dimeric WT PH domain possesses a one order of magnitude higher target membrane affinity than the previously characterized monomeric PH domains, consistent with a dimerization-triggered, allosterically enhanced affinity for one PIP₃ molecule (a much larger affinity enhancement would be expected for dimerization-triggered binding to two PIP₃ molecules). The monomeric T513E PDK1 PH domain, like other monomeric PH domains, exhibits a PIP₃ affinity and bound state lifetime that are each 1 order of magnitude lower than those of the dimeric WT PH domain, which is predicted to facilitate release of activated, monomeric PDK1 to the cytoplasm. Overall, the study yields the first molecular picture of PH domain regulation via electrostatic control of dimer鈥搈onomer conversion.