Identification and characterisation of proteins associating with the collagen homology domains of p66Shc

The Src-homology collagen protein (Shc) is expressed as three isoforms of 46, 52 and 66 kDa. The 66 kDa isoform of Shc (p66Shc) has been reported to inhibit the MAPK pathway and to sensitive cells to oxidative stress. To investigate the unique characteristics of the p66Shc isoform, it was attempted to identify proteins that associate with the second collagen homology domain (CH2). Using the yeast two-hybrid system, the receptor tryrosine phosphatase Lar and the colonic and hepatic tumour over expressed gene Ch-Tog were identified as binding partners for the CH2 domain of p66Shc. These observations were verified through co-immunoprecipitation and GST fusion protein precipitations. A common motif of SSKXXQ was identified in both Lar and Ch-Tog. Mutational analysis of this motif in Lar demonstrates that lysine 1285 of human Lar is essential for its association with p66Shc, as demonstrated with the yeast two-hybrid system, co-immunoprecipitation and GST fusion protein precipitation experiments. Although p66Shc is constitutively associated with the Lar tyrosine phosphatase, it is not, however a substrate of Lar. Ch-Tog is a component of the spindle checkpoint. Activators of the spindle checkpoint such as the anticancer drug Taxol induce a substantial serine phosphorylation of p66Shc. p66Shc is required for the efficient apoptosis of mouse embryo fibroblast cells as cells lacking p66Shc show considerable resistance to Taxol even at high does (1mM). Taxol induced serine phosphorylation of p66Shc is an M phase specific event that is independent of microtubule condition. Jnk, p38 and Erk activation are not involved in the resistance of p66Shc knockout cells to Taxol. The Cyclin dependent kinase inhibitor Roscovitine completely inhibits the serine phosphorylation of p66Shc after Taxol treatment. In gel kinase assays have identified five kinases capable of phosphorylating the CH2 domain of p66Shc, but their identity is not yet known. Jnk, p38, Cdk1, Erk1 and Erk2 do not phosphorylate the CH2 domain of p66Shc in vitro..