Casein Kinase Substrates 3

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

Objective: Machado-Joseph disease (MJD)/Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 (CK2). Methods: The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase (GST) pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results (1) Both wild type and expanded ataxin-3 interacted with CK2alpha and CK2beta in vitro. (2) In 293 cells, both wild type and expanded ataxin-3 interacted with CK2beta, but not CK2alpha. (3) CK2 phosphorylated wild type and expanded ataxin-3. Conclusion: Ataxin-3 is a substrate of protein kinase CK2.

We have previously shown that casein kinase (CK) Ialpha from mammalian brain phosphorylates 14-3-3 zeta and tau isoforms on residue 233. In the present study, we show that CKIalpha associates with 14-3-3 both in vitro and in vivo. The interaction between CKIalpha and 14-3-3 is dependent on CKIalpha phosphorylation, unlike centaurin-alpha1 (also known as ADAP1), which binds to unphosphorylated CKIalpha on the same region. CKIalpha preferentially interacts with mammalian eta and gamma 14-3-3 isoforms, and peptides that bind to the 14-3-3 binding pocket prevent this interaction. The region containing Ser218 in this CKIalpha binding site was mutated and the interaction between CKIalpha and 14-3-3 was reduced. We subsequently identified a second phosphorylation-dependent 14-3-3 binding site within CKIalpha containing Ser242 that may be the principal site of interaction. We also show that both fission and budding yeast CKI kinase homologues phosphorylate mammalian and budding yeast (BMH1 and BMH2) 14-3-3 at the equivalent site.