Coilin phosphorylation mediates interaction with SMN and SmB′
- 236 Downloads
Cajal bodies (CBs) are subnuclear domains that participate in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and play a part in the assembly of the spliceosomal complex. The CB marker protein, coilin, interacts with survival of motor neuron (SMN) and Sm proteins. Several coilin phosphoresidues have been identified by mass spectrometric analysis. Phosphorylation of coilin affects its self-interaction and localization in the nucleus. We hypothesize that coilin phosphorylation also impacts its binding to SMN and Sm proteins. In vitro binding studies with a C-terminal fragment of coilin and corresponding phosphomimics show that SMN binds preferentially to dephosphorylated analogs and that SmB′ binds preferentially to phosphomimetic constructs. Bacterially expressed full-length coilin binds more SMN and SmB′ than does the C-terminal fragment. Co-immunoprecipitation and phosphatase experiments show that SMN also binds dephosphorylated coilin in vivo. These data show that phosphorylation of coilin influences interaction with its target proteins and, thus, may be significant in managing the flow of snRNPs through the CB.
KeywordsNormal Mouse Serum C214 Fragment Immunoprecipitation Reaction Subnuclear Domain HeLa Lysate
This work was supported by NIH grant R01GM081448 to MDH. We thank Dr. Angus Lamond (University of Dundee, Dundee, Scotland) for his gift of the YFP-SmB′ HeLa stable line.
- Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villen J, Li J, Cohn M.A, Cantley LC, Gygi SP (2004) Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A. 101:12130–12135Google Scholar
- Dephoure N, Zhou C, Villen J, Beausoleil SA, Bekalarski CE, Elledge SJ, Gygi SP (2008) A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A. 105:10762–10767Google Scholar
- Nousiainen M, Sillje HH, Sauer G, Nigg EA, Korner R (2006). Phosphoproteome analysis of the human mitotic spindle. Proc Natl Acad Sci U S A. 103:5391–5396Google Scholar
- Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P, Mann M (2006) Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell. 127:635–648Google Scholar
- Sleeman J, Lyon CE, Platani M, Kreivi J-P, Lamond AI (1998) Dynamic interactions between splicing snRNPs, coiled bodies and nucleoli revealed using snRNP protein fusions to the green fluorescent protein. Exp. Cell Res. 243:290–304Google Scholar