Sorting nexin, SH3PX1, has been associated with endocytosis through its interactions with key endocytic proteins involved in the processing of cell surface receptors. These dramatic cellular effects, largely associated with the tyrosine phosphorylation of SH3PX1, have elicited interest in determining the molecular mechanisms underlying this phosphorylation event.

The first major focus of these studies was to develop reagents that could be used to further characterize the role of SH3PX1 in endocytosis. SH3PX1 has been identified as a substrate of the nonreceptor tyrosine kinase, activated Cdc42-associated kinase-2 (ACK2), and the resulting phosphorylation enhances degradation of the epidermal growth factor (EGF) receptor in cells. In order to further characterize ACK2-catalyzed SH3PX1 phosphorylation, we have identified tyrosine 287 as a major site of phosphorylation by mass spectrometry. Moreover, we have shown that the pyridopyrimidine PD158780 is a potent inhibitor of ACK2 kinase activity in vitro (IC50 ~80 pM). Together, we believe that phosphorylation-defective mutants of SH3PX1 and small molecule inhibitors of ACK2 kinase activity will help to further establish the roles of ACK2 and SH3PX1 in EGF receptor processing.

The second aspect of these studies involved the identification and characterization of novel tyrosine kinases that phosphorylate SH3PX1 in cells, namely focal adhesion kinase (FAK) and Src. Here, we show that FAK and Src differ considerably from ACK2 in their abilities to phosphorylate SH3PX1. For example, FAK and Src are more effective kinases for SH3PX1, compared to ACK2. In addition, we show that FAK and Src are able to phosphorylate several carboxyl-terminal truncation mutants of SH3PX1 that are defective for ACK2-catalyzed phosphorylation, suggesting that FAK and Src bind to and phosphorylate different sites on SH3PX1. This was further confirmed by mass spectrometry analysis which identified residues Y177, Y239, Y269, Y294, and Y561, as Src-catalyzed phosphorylation sites, with Y239 as the major site. Given the observed differences exhibited by FAK and Src, versus ACK2, in binding and phosphorylating SH3PX1, it now seems likely that this sorting nexin may be responsible for translating a complicated array of regulatory inputs into the endocytosis and degradation of membrane receptors.