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SR proteins are a family of splicing factors that plays important roles in regulating alternative splicing. The function of SR proteins is heavily regulated by the extent of phosphorylation of its C-terminal RS domain. Cdc-2 like kinases (CLKs) are known to hyper-phosphorylate the RS domains and control the splicing functions of SR proteins. CLKs have a folded kinase domain and an N-terminus that is predicted to be disordered. The N-terminus plays vital roles in regulating the function of CLK1 in various aspects. The studies presented here elucidate how the N-terminus modulates three different aspects of CLK1 function: sub-cellular localization, self-association to form oligomers, and substrate phosphorylation. Although prior studies had shown that the N-terminus was important for modulating the nuclear import of CLK1, the mechanism for this change in subcellular localization had largely been uninvestigated. Here, we show that CLK1 lacks a short, classical Nuclear Localization Sequence (NLS), indicating that the nuclear import is not mediated by the classical importin / system. Instead, we show that CLK1 enters the nucleus by forming a complex with its physiological substrate SRSF1, an SR protein prototype, in the cytoplasm and transportin SR-2 (TRN-SR2) imports the kinase-substrate complex into the nucleus. Previous studies from our laboratory had shown that the N-terminus induces oligomerization of CLK1, which helps the kinase select its physiological substrates over non-physiological ones. The nature of the interactions underlying this oligomerization was investigated and our results show that CLK1 oligomerization is driven not only by self-association of the N-terminus (N-N interactions) but also by interactions between the N-terminus and the kinase domain (N-K interactions). While interactions between the N-termini are mediated solely by aromatic residues, interactions between the N-terminus and kinase domain are electrostatic in nature. Lastly, we also inves

SR proteins are a family of splicing factors that plays important roles in regulating alternative splicing. The function of SR proteins is heavily regulated by the extent of phosphorylation of its C-terminal RS domain. Cdc-2 like kinases (CLKs) are known to hyper-phosphorylate the RS domains and control the splicing functions of SR proteins. CLKs have a folded kinase domain and an N-terminus that is predicted to be disordered. The N-terminus plays vital roles in regulating the function of CLK1 in various aspects. The studies presented here elucidate how the N-terminus modulates three different aspects of CLK1 function: sub-cellular localization, self-association to form oligomers, and substrate phosphorylation. Although prior studies had shown that the N-terminus was important for modulating the nuclear import of CLK1, the mechanism for this change in subcellular localization had largely been uninvestigated. Here, we show that CLK1 lacks a short, classical Nuclear Localization Sequence (NLS), indicating that the nuclear import is not mediated by the classical importin / system. Instead, we show that CLK1 enters the nucleus by forming a complex with its physiological substrate SRSF1, an SR protein prototype, in the cytoplasm and transportin SR-2 (TRN-SR2) imports the kinase-substrate complex into the nucleus. Previous studies from our laboratory had shown that the N-terminus induces oligomerization of CLK1, which helps the kinase select its physiological substrates over non-physiological ones. The nature of the interactions underlying this oligomerization was investigated and our results show that CLK1 oligomerization is driven not only by self-association of the N-terminus (N-N interactions) but also by interactions between the N-terminus and the kinase domain (N-K interactions). While interactions between the N-termini are mediated solely by aromatic residues, interactions between the N-terminus and kinase domain are electrostatic in nature. Lastly, we also inves

SR proteins are a family of splicing factors that plays important roles in regulating alternative splicing. The function of SR proteins is heavily regulated by the extent of phosphorylation of its C-terminal RS domain. Cdc-2 like kinases (CLKs) are known to hyper-phosphorylate the RS domains and control the splicing functions of SR proteins. CLKs have a folded kinase domain and an N-terminus that is predicted to be disordered. The N-terminus plays vital roles in regulating the function of CLK1 in various aspects. The studies presented here elucidate how the N-terminus modulates three different aspects of CLK1 function: sub-cellular localization, self-association to form oligomers, and substrate phosphorylation. Although prior studies had shown that the N-terminus was important for modulating the nuclear import of CLK1, the mechanism for this change in subcellular localization had largely been uninvestigated. Here, we show that CLK1 lacks a short, classical Nuclear Localization Sequence (NLS), indicating that the nuclear import is not mediated by the classical importin / system. Instead, we show that CLK1 enters the nucleus by forming a complex with its physiological substrate SRSF1, an SR protein prototype, in the cytoplasm and transportin SR-2 (TRN-SR2) imports the kinase-substrate complex into the nucleus. Previous studies from our laboratory had shown that the N-terminus induces oligomerization of CLK1, which helps the kinase select its physiological substrates over non-physiological ones. The nature of the interactions underlying this oligomerization was investigated and our results show that CLK1 oligomerization is driven not only by self-association of the N-terminus (N-N interactions) but also by interactions between the N-terminus and the kinase domain (N-K interactions). While interactions between the N-termini are mediated solely by aromatic residues, interactions between the N-terminus and kinase domain are electrostatic in nature. Lastly, we also inves

This thesis details efforts to generate chemical probes for the interrogation of important protein targets: the CLK and DYRK kinases, ceramide synthases (CerS), and the PP2A-inhibitory protein, SBDS. Modulation of activity with chemical probes and protein degradation with PROTACs were investigated. Chapter 1 provides an introduction to chemical probes and PROTACs, introduces the relevant protein targets, and provides background on previous work by the Morris group. Chapters 2-4 are focused on the development of chemical probes and PROTACs for the CLK and DYRK kinases. In Chapter 2, an analysis of crystal structures and inhibitors of CLK1 and DYRK1a resulted in elucidation of the factors that determine selective inhibition. This analysis led to the investigation of 6-azaindole 2.3—previously reported as a DYRK1a-selective inhibitor— and it was found to be a potent inhibitor of CLK1, CLK2, and DYRK1a. Insights gained from the structural analysis were used to design quinoline 2.36, a CLK1 inhibitor with 15-fold selectivity over DYRK1a.Work in Chapter 3 led to improved pharmacophore models for selectivity and potency, which were used to develop novel potent CLK/DYRK inhibitors, including 3-quinoline 3.44, a 1.2 nM inhibitor of DYRK1a with 5.4-fold selectivity over CLK1. Chapter 4 describes a preliminary study of 6-azaindole PROTACs targeted towards CLK1/DYRK1a. A small library of PROTACs based on amide 4.5 were synthesised and initial biological evaluation by our collaborators did not indicate degradation of CLK1 or DYRK1a at 1 µM across multiple treatment times, possibly due to insufficient formation of stable ternary complexes. Chapter 5 outlined investigations with the AAL(S) scaffold. A preliminary investigation of PROTACs for the degradation of CerSs was carried out, with a small library of CerS PROTACs synthesised. These influenced cellular ceramide concentrations, via a yet-to-be-determined mechanism. The second part of Chapter 5 was focused on the elucidation of