The Csk-homologous kinase CHK is a potential colorectal cancer (CRC) tumour suppressor of which the expression is down-regulated by epigenetic silencing. It is well known that CHK exerts its tumour suppressive action in part by phosphorylating and inhibiting the Src-family protein tyrosine kinases (SFKs), which are over-activated in CRC cells. Being a protein kinase, CHK can potentially exert its tumour suppressive action by phosphorylating other non-SFK protein substrates. In this study, the tumour suppressive mechanism of CHK is investigated by incorporating two phosphopeptide enrichment approaches to analyse the phosphoproteome of the transduced CRC DLD1 cells capable of expressing recombinant CHK upon induction by doxycycline. The first approach, capable of identifying and quantifying the abundance of mainly phospho-serine- and phosphothreonine-containing peptides, employs a label-free proteomic method coupled with enrichment of phosphopeptides by titanium dioxide-affinity purification. To determine the changes of phospho-tyrosine (pY)-containing proteome of the transduced DLD1 cells in response to induced expression of recombinant CHK, we adapted the use the mutant SH2 domains (referred to as SH2 pY-superbinders) with significantly enhanced affinities for pY-containing proteins and peptides to enrich pY-containing peptides for proteomic analysis. In the second approach, perturbation of the pY proteome was accessed by the isotopic dimethyl labelled phosphoproteomic method coupled with the use of SH2 pY-superbinders. The combined results from both approaches revealed for the first time activation of the mitogen-associated protein (MAP) kinase signaling pathway upon CHK induction in CRC cells. Furthermore, the second approach identified several cellular proteins such as the membrane-bound myelin protein zero-like protein 1 (PZR) as potential non-SFK substrates of CHK. Further bioinformatic analysis suggests that its enhanced phosphorylation mediates activation of the MAP kinase signaling pathway induced by expression of recombinant CHK. The analysis also suggests that the CHK/PZR/MAP kinase pathway mediates the effect of CHK to cause cell senescence of CRC cells.