Molecular and Functional Analysis of Phosphatidylinositol 4 Kinase Type II Beta




Jung, Gwanghyun

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Phosphoinositides play fundamental roles in controlling membrane-based signaling events. Phosphatidylinositol 4-kinases (PI4Ks) catalyze the production of PI4P, a major precursor in phosphoinositide biosynthesis, and consist of two classes (type II and type III), each divided into two isoforms (alpha and beta ). PI4KIIalpha and beta differ primarily in their distributions between cytosol and membranes: PI4KIIalpha is almost exclusively membrane-bound, by virtue of its palmitoylation in a cysteine-rich motif; although PI4KIIbeta also contains a palmitoylatable cysteine-rich motif, this isoform is almost evenly distributed between membranes and cytosol, and only about half of the membrane-associated pool is palmitoylated.
My study focused on determining the functions of post-translational modifications and domains of PI4KIIbeta and on identifying its binding partners, with the long-term goal of understanding the roles and mechanisms of regulation of this kinase. Domain analysis shows that the C-terminal 160 amino acids of PI4KIIs determine the distinct membrane binding properties and activities of PI4KIIalpha and beta . As expected, based on our previous work with PI4KIIalpha , palmitoylation of PI4KIIbeta is important for its membrane binding and activity. Although PI4KIIbeta is also phosphorylated in cells, this modification has no detectable effect on any examined property of the kinase.
Immunoprecipitation and mass spectrometry revealed a genuine binding partner for PI4KIIbeta , Hsp90. The functional significance of the Hsp90-PI4KIIbeta interaction was defined using geldanamycin, a specific Hsp90 inhibitor. Geldanamycin treatment disrupts the interaction and destabilizes PI4KIIbeta , reducing its half-life by 40% and increasing its susceptibility to proteasomal degradation. Although full-length PI4KIIalpha does not bind Hsp90, and is not destabilized by geldanamycin treatment, a cytosolic PI4KIIalpha truncation mutant becomes sensitive to geldanamycin and binds to Hsp90. Thus, both PI4KII isoforms contain Hsp90 binding sites but only PI4KIIbeta requires Hsp90 for stabilization, presumably because there is a substantial cytosolic pool of this isoform. Interestingly, brief exposure to geldanamycin causes a partial redistribution of PI4KIIbeta from the cytosol to membranes, which results in increased PI4P synthesis in cells. Moreover, the growth factors EGF and PDGF also disrupt the interaction between Hsp90 and PI4KIIbeta , suggesting that Hsp90 not only protects PI4KIIbeta from degradation, but may also prolong its residency in the cytoplasm until extracellular signals release Hsp90 from the kinase.
Currently the precise roles of PI4KIIbeta are unknown. Based on its partial redistribution to the plasma membrane upon cell treatment with growth factors, I speculated that PI4KIIbeta may somehow be involved in receptor-mediated endocytic trafficking. My results, employing siRNA-based knockdown strategies, indicate that depletion of PI4KIIbeta enhances early steps of EGFR internalization and subsequent initiation of ERK activation in response to EGF treatment. The facilitated endocytosis that results from this depletion is likely due to an increase in endosomal fusion. Indeed, activities of the endosomal fusion facilitators EEA1 and Rab5 increase in PI4KIIbeta depleted cells. These results suggest for the first time a role of PI4KIIbeta in endocytic trafficking and signaling of the EGFR.

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