Web Release Date: October 18,
Novel Destabilization of Nucleotide Binding by the 
Phosphate of ATP in the
Yeast SR Protein Kinase Sky1p
and
Departments of Pharmacology and Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0506
Received July 9, 2003
Revised Manuscript Received August 22, 2003
Abstract:
SR protein kinases (SRPKs) regulate the temporal and cell-specific selection of alternative
splice sites. These enzymes are highly unique members of the protein kinase family. SRPKs contain a
large domain insert (approximately 200 residues) within the kinase core, do not require phosphorylation
for regulation, have an extended helix insert near the nucleotide pocket, and possess unusual substrate
specificity determinants. The yeast SRPK, Sky1p, rapidly phosphorylates its natural substrate Npl3 but
binds ATP with a high Km, suggesting that some of these distinctive structural features may be correlated
with nucleotide binding [Aubol et al. (2002) Biochemistry 41, 10002-10009]. To address this issue, the
nucleotide binding properties of Sky1p were studied using fluorescence spectroscopy. The affinities of
several nucleotides (ATP, ADP, AMP, adenosine, and AMPPNP) to Sky1p and the prototype kinase,
cAMP-dependent protein kinase, were compared in the absence and presence of the metal activator, Mg2+,
using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent
protein kinase, potently destabilizes the phosphate of ATP. This novel finding suggests that rapid
phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.
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