Interactions between Casein kinase Iepsilon (CKIepsilon) and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.
BACKGROUND
Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.


RESULTS
CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.


CONCLUSIONS
The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.
Dahlberg
Caroline Lund
CL
Department of Biochemistry, University of Washington, Seattle, Washington, United States of America.
Nguyen
Elizabeth Z
EZ
Goodlett
David
D
Kimelman
David
D
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D048149:Casein Kinase 1 epsilon / Q000378:metabolism*; D048128:Casein Kinase Ialpha / Q000378:metabolism; D000072261:Dishevelled Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D056950:Period Circadian Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D015398:Signal Transduction; D013379:Substrate Specificity; D014981:Xenopus; D029867:Xenopus Proteins
D048868:Adaptor Proteins, Signal Transducing; D000072261:Dishevelled Proteins; D047908:Intracellular Signaling Peptides and Proteins; C539025:Per1 protein, Xenopus; C539023:Per1 protein, mouse; D056950:Period Circadian Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D029867:Xenopus Proteins; D048149:Casein Kinase 1 epsilon; D048128:Casein Kinase Ialpha