Abstract
A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.
Highlights
A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed
We identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A
Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domaincontaining proteins
Summary
Screen in parallel for interaction partners and substrates of the kinase DYRK1A. In this approach, we used an arrayed protein expression library [29, 30] both for interaction screening by overlaying with the radiolabeled recombinant kinase [31], and for a substrate screen by solid phase phosphorylation [26]. Several DYRK1A-interacting and/or phosphorylated proteins were identified by this approach, and one protein was found in both screens. This protein turned out to be a novel member of the cyclin family and was designated cyclin L2. We characterize cyclin L2 as a novel substrate of DYRK1A. We demonstrate that cyclin L2 and the closely related isoform, cyclin L1, differ strikingly with regard to their intranuclear mobility
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