Novel destabilization of nucleotide binding by the gamma phosphate of ATP in the yeast SR protein kinase Sky1p.
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 K(m), 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, Mg(2+), using a fluorescence-based displacement assay. The data indicate that Sky1p, unlike cAMP-dependent protein kinase, potently destabilizes the gamma phosphate of ATP. This novel finding suggests that rapid phosphoryl transfer may be facilitated by unique mechanisms in both protein kinases.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0506, USA.
Brandon E
BE
Aubol
Brad
B
Nolen
Jennifer
J
Shaffer
Gourisankar
G
Ghosh
Joseph A
JA
Adams
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry*; D001665:Binding Sites / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D004579:Electron Transport / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D016254:Mutagenesis, Insertional; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity / Q000235:genetics; D062367:ortho-Aminobenzoates / Q000737:chemistry*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D062367:ortho-Aminobenzoates; D000244:Adenosine Diphosphate; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate; C490216:SKY1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium