Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain: phosphorylation by DYRK1A and colocalization with splicing factors.
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.
de Graaf
Katrin
K
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen, Wendlingweg 2, 52074 Aachen, Germany, Institut für Biochemie, Medizinische Fakultät der RWTH Aachen, Pauwelstrasse 30, 52074 Aachen, Germany.
Hekerman
Paul
P
Spelten
Oliver
O
Herrmann
Andreas
A
Packman
Len C
LC
Büssow
Konrad
K
Müller-Newen
Gerhard
G
Becker
Walter
W
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D016213:Cyclins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D014157:Transcription Factors; D000097603:Dyrk Kinases
D001120:Arginine; D016213:Cyclins; D018076:DNA, Complementary; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012694:Serine; D014157:Transcription Factors; D000097603:Dyrk Kinases; C483080:CCNL2 protein, human