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J. Cell Biol.,
Volume 145, Number 3, May 3, 1999 447-455
* Division of Cellular and Molecular Medicine, Department and School of Medicine, University of California, San Diego, La
Jolla, California 92093-0651; and The SR superfamily of splicing factors and
regulators is characterized by arginine/serine (RS)-rich
domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS
domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of
the single nonessential SR protein-specific kinase
Sky1p in Saccharomyces cerevisiae, we investigated RS
domain interactions in vivo using the two-hybrid assay.
Strikingly, all RS domain-mediated interactions were
abolished by SKY1 deletion and were rescuable by
yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on
RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization
of SR proteins and found that SC35 was shifted to the
cytoplasm in sky1 Department of Molecular and Cell Biology, Harvard University, Cambridge, Massachusetts
02138
yeast, although this phenomenon
was not obvious with ASF/SF2, indicating that nuclear
import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression
assay, we further showed that most LexA-SR fusion
proteins depend on Sky1p to efficiently recognize the
LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within
the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR
proteins to interact with one another efficiently and
specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.
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