Biochemistry and Pharmacology - Theses

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    Mechanism of action of the potential tumour suppressor Csk homologous kinase (Chk) in colorectal cancer cells
    Advani, Gahana Ravi ( 2018)
    Aberrant activation of Src family tyrosine kinases (SFKs) directs initiation of metastasis and development of drug resistance in multiple solid tumours and haematological cancers. Intriguingly, 80% of colorectal cancer (CRC) cells exhibit a 5 to 10-fold increase in SFK activity directly correlating with their oncogenic potential. Since oncogenic mutations of SFKs are rare events, aberrant activation of SFKs in cancer is likely due to dysregulation of the two major upstream inhibitors: C-terminal Src kinase (Csk) and its homolog Csk homologous kinase (Chk). SFKs are maintained in the inactive conformation by two major intermolecular interactions (i) binding of the SH3 domain to the SH2-kinase linker and (ii) binding of the C-terminal tail phosphotyrosine to the SH2 domain. Phosphorylation of the C-terminal tail tyrosine is a pre-requisite for SFKs to adopt this closed inactive conformation. Both Csk and Chk can phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Moreover, they can also employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types, suggesting that they perform overlapping as well as distinct functions to suppress SFK activity in cells. My PhD project aimed to delineate how Csk and Chk cooperate to inhibit SFK activity in cells. Specifically, I define (i) the contributions of the catalytic and non-catalytic inhibitory mechanisms towards the inhibitory activity of Csk and Chk and (ii) the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism. Using biochemical and biophysical approaches, I determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced CRC cells. Specifically, I determined the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and the SFK member, Src. We employed Surface plasmon resonance (SPR) spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of SFK. Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of phosphorylation of the SFK C-terminal tail tyrosine but it binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism. Next, I sought to determine the functional domain in Chk responsible for binding and inhibition of SFKs. Using an engineered Csk-Chk chimera and SPR, I mapped the major determinants in Chk governing its tight binding with Src to its kinase domain. Previous studies of the crystal structure of the Csk kinase domain in complex with Src revealed five basic arginine residues responsible for Csk binding to Src; mutations of any one of these basic residues significantly reduced the affinity of binding of Csk to Src and abolished the inhibitory activity of Csk1. Since these residues are conserved in the Chk kinase domain, we hypothesized that they are also determinants governing Chk binding and inhibition of SFKs. Studies of Chk mutants generated by site-directed mutagenesis revealed that mutations of two of the five arginine residues in Chk had a slight impact on its binding affinity to the SFK. However, the mutations did not impact the ability of Chk to inhibit SFKs by the non-catalytic mechanism. Based upon these findings, we propose a model in which the optimal alignment of the five basic residues in Csk and Chk is critical for their inhibition of SFKs. The model also suggests that the Chk kinase domain exhibits a much higher propensity than the Csk kinase domain to adopt this configuration. Chk can efficiently employ the non-catalytic inhibitory mechanism to inhibit multiple active forms of SFKs as compared to Csk, suggesting that Chk is a versatile tumour suppressor capable of constraining the activity of multiple active forms of SFKs. The lack of this non-catalytic inhibitory mechanism may account for SFK overactivation in the Chk-deficient CRC cells. Additionally, several pieces of evidence suggest Chk as a potential tumour suppressor down-regulated by epigenetic silencing and/or inactivated by missense mutations in several cancer cases such as colorectal and lung carcinoma. In spite of the potential significance of Chk in cancer development, convincing evidence supporting Chk as a tumour suppressor has not been presented. Our collaborators and us previously reported down-regulation of Chk expression in several CRC cell lines and in CRC biopsies obtained from several patients2, suggesting that Chk is a potential colorectal tumour suppressor and suppression of its expression contributes to cancer initiation and/or progression. In my PhD study, I conducted experiments to further confirm that Chk is a potential CRC tumour suppressor and aimed to delineate its tumour suppressive mechanism in CRC cells. Specifically, I used transduced human CRC cells expressing recombinant Chk under the control doxycycline as the model system and examined how recombinant Chk expression affects the oncogenic phenotypes and SFK activity in these cells. First, q-PCR further confirmed downregulation of Chk in a panel of CRC cell lines and that Chk expression is likely suppressed at the transcriptional level. To ascertain if Chk gene transcription is suppressed by epigenetic silencing, I performed genome-wide methylation and bisulfite sequencing studies and revealed hypermethylation of the Chk gene promoter in a panel of CRC cell lines. Our collaborators in the Walter and Eliza Hall Institute in Melbourne also discovered that treatment with a panel of CRC cell lines with inhibitors of epigenetic silencing led to an increase in the mRNA level of Chk. Collectively, these results strongly suggest that expression of Chk is down-regulated in CRC cells by an epigenetic silencing mechanism involving DNA methylation of the Chk gene promoter. Additionally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient CRC cells. Reintroduction of Chk in CRC cell lines resulted in robust inhibition of Src activity and suppression of anchorage-independent growth. As Chk is a protein kinase directly phosphorylating multiple substrates in cells, it may exert its tumour suppressive action by phosphorylating SFKs as well as other cellular proteins. Similar to protein kinases containing protein-protein interaction domains such as SH2 and SH3 domains, Chk may use its SH2 and/or SH3 domains to bind its non-SFK substrates prior to their phosphorylation. As an attempt to search for and identify these non-SFK substrates of Chk in transduced CRC cells, I used the DLD1-Chk-GFP expressing CRC cell line and an unbiased proteomics approach to identify the cellular proteins bound to recombinant Chk-GFP in the transduced DLD-1 cells. My analysis revealed co-immunoprecipitation of Chk-GFP with Src and many other non-SFK proteins. Besides acting as the direct substrates, some of the non-SFK cellular proteins bound to Chk may also be upstream regulators that bind and control Chk functions in CRC cells. Thus, further investigation focusing on these Chk-binding proteins will identify the potential upstream regulators and direct protein substrates of Chk. In summary, my findings strongly suggest that Chk is potential tumour suppressor inhibiting SFK activity by the non-catalytic inhibitory mechanism. Specifically, my research results provide biochemical and structural insights into the mechanism of the tumour suppressive action and regulation of Chk in CRC cells. Future studies to clearly define the molecular basis of the tumour suppressive action of Chk may benefit the development of new therapeutic strategies for the treatment of CRC.