No abstract

The kinesin motors are important in the regulation of cellular functions such as protein trafficking, spindle organization and centrosome separation. In this study, we have identified POPX2, a serine-threonine phosphatase, as an interacting partner of the KAP3 subunit of the kinesin-2 motor. The kinesin-2 motor is a heterotrimeric complex composed of KIF3A, KIF3B motor subunits and KAP3, the non-motor subunit, which binds the cargo. Here we report that the phosphatase POPX2 is a negative regulator of the trafficking of N-cadherin and other cargoes; consequently, it markedly influences cell-cell adhesion. POPX2 affects trafficking by determining the phosphorylation status of KIF3A at serine 690. This is consistent with the observation that the KIF3A-S690A mutant is defective in cargo trafficking. Our studies also implicate CaMKII as the kinase that phosphorylates KIF3A at serine 690. These results strongly suggest that POPX2 and CaMKII are a phosphatase-kinase pair that regulates kinesin-mediated transport and cell-cell adhesion.

Identifying the substrates and biochemical pathway regulated by phosphatases has always been more challenging than finding those regulated by kinases. Here, we report the use of phosphoproteomic methods to analyse the pathways regulated by POPX2 (partner of PIX 2) phosphatase. POPX2 is a serine/threonine phosphatase, found in many cancer types. The levels of the POPX2 have been found to be up‐regulated in the more invasive breast cancer cells compared with non‐invasive ones. Our observations also suggest that POPX2 level is positively correlated with cell motility. Thus, finding substrates or pathways regulated by POPX2 will help to elucidate the regulatory mechanism of cancer cell motility and invasiveness. We have also developed and validated a protocol using electrostatic repulsion‐hydrophilic interaction chromatography (ERLIC) to enrich the phosphopeptides followed by LC‐MS/MS to allow comparison between the phosphoproteomes of control and POPX2 overexpressing cells. With this approach, we were able to identify a biochemical pathway through which POPX2 exerts its apparent cellular function: the regulation of activity of glycogen synthase kinase‐3, which in turn modulates extracellular signal‐regulated kinase and cell motility.