PDK1 PARTICIPATES IN THE CHAPERONE-MEDIATED RESCUE OF THE POLARITY COMPLEX ATYPICAL PKC IN INTESTINAL EPITHELIA.

Atrium

Objective. Atypical protein kinase C (aPKC) has a well-established role in the development of epithelial polarity. This study was conducted to determine the identity of the kinase responsible for rephosphorylation of aPKC activation domain after chaperone-mediated rescue from degradation. Background. To become active and fully functional in the cell all PKC family members need to acquire a special active conformation, which is achieved through phosphorylation. This phosphorylation is necessary not only for newly synthesized molecules, but also for kinase molecules that become dephosphorylated and need to be refolded and rephosphorylated. This “rescue” mechanism is responsible for the maintenance of the steady-state levels of aPKC. There is consensus that phosphoinositide-dependent protein kinase 1 (PDK1) is the activating kinase for newly synthesized PKC molecules. 60 We have hypothesized that PDK1 is involved in rescue rephosphorylation in addition to its role in activating newly synthesized aPKC. Methods. To test our hypothesis we inhibited protein synthesis and analyzed the stability of the remaining aPKC pool in the cells where function of PDK1 was abolished with specific inhibitors or by shRNAmediated knockdown. Results. PDK1 knockdown and application of two different PDK1 inhibitors destabilized the pool of active aPKC. PDK1 coimmunoprecipitated with aPKC in cells without protein synthesis, confirming that the interaction is direct. In addition, we showed that PDK1 aids the rescue of aPKC in in vitro rephosphorylation assays. Conclusion. PDK1 is the kinase that rephosphorylates aPKC after chaperone-mediated rescue and refolding in polarized epithelial cells. Grants. This work was supported by NIH Award R01DK087359 to Pedro Salas.