| Popis: |
The small GTPase Rab11 family proteins have been implicated in the plasma membrane recycling system in such diverse model systems as H/K-ATPase trafficking in parietal cells to GLUT4 trafficking in heart and skeletal muscle. Rab11 family interacting protein 2 (Rab11-FIP2) was previously identified as binding to both Rab11a and the motor protein, myosin Vb. Therefore, we hypothesized that Rab11-FIP2 is a critical regulator of the plasma membrane recycling system. In these studies, we sought to characterize the role of Rab11-FIP2 function in polarized epithelial cells. To address the role of Rab11-FIP2, we uncovered a new role for Rab11-FIP2 in the establishment of polarity. We found that Rab11-FIP2 is phosphorylated by MARK2, and that this phosphorylation is necessary for the proper formation of the adherens junction. Next, we characterized a new dominant mutant Rab11-FIP2 mutant, which has unique effects on the recycling system than all previously characterized mutants. Analysis of the data suggests that FIP2 is involved in multiple stages in passage through the Rab11a associated recycling system. Multiple points of entry into the Rab11a/ FIP2 recycling system may be exploited depending upon the origin of the protein and possibly, its destination. This model supports a dynamic vision of the recycling system trafficking. We also analyzed new Rab11-FIP2 interacting proteins utilizing a novel approach of immunoprecipitation from stable cell lines overexpressing either wild type Rab11-FIP2 or its mutants followed by identification of the associated proteins using mass spectrometry. This proteomics approach revealed novel interactions with proteins known to be involved in trafficking (dynein and Rab10) in early endosomal membrane regulation (Rab5b and EpsinR), and vesicle coat proteins (AP-1 and clathrin heavy chain). Validation of these interactions proves that this methodology reveals robust interactions that are readily confirmed. All together, this body of work significantly advances our understanding of the diverse roles of Rab11-FIP2 in the regulation of epithelial cell function. |
