Project Summary Dual specificity protein kinases perform indispensable roles in eukaryotes including the regulation of signal transduction pathways and subcellular compartmentalization. Structurally, in addition to their folded kinase domains, they are composed of significantly long segments that are intrinsically disordered. Whereas the folded domains of these kinases are studied extensively (both biochemically and structurally), little is known about the functional role of the disordered domains that display a compositional bias towards polar and charged amino acids. In our preliminary sequence analysis of all human dual specificity kinases, we identify that CLK3 has the longest intrinsically disordered region. Recent advances indicate that CLK3 is linked to multiple cancer pathologies and celiac disorders. For example, overexpression of CLK3 is associated with osteosarcoma, liver cancer and bile cancer. Sequence variations in CLK3 gene is associated with bladder cancer, Crohn’s disease and multiple celiac disorders. Functionally, CLK3 is recruited to nuclear speckles and plays vital roles in the regulation of pre-mRNA splicing. In this grant application, we propose to characterize the sequence determinants, molecular codes and functional roles of CLK3 nuclear speckle recruitment utilizing a combination of in vitro and in vivo experiments. Based on our preliminary analyses, we posit that (i) the low-complexity disordered domain of CLK3 (residue 1-285) drives the protein’s nuclear speckle localization, and (ii) alterations in CLK3 speckle localization/dynamics is associated with disease pathologies. We will test these hypotheses utilizing an integrative biophysical, biochemical and cell biology approach. To this end, we will utilize a splice variant, a disease-linked mutant, and rationally perturbed LCD variants to systematically decipher the LCD’s role in CLK3’s function/dysfunction. Successful completion of the proposed study will not only illuminate the role of this disordered domain in CLK3 (patho)biology, but also provide significant insights into the interplay between the LCDs and kinase domains in dual specificity kinase functions and dysfunctions.