Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1.
The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism--a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein-protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.
Hagopian
Jonathan C
JC
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0636, USA.
Ma
Chen-Ting
CT
Meade
Bryan R
BR
Albuquerque
Claudio P
CP
Ngo
Jacky Chi Ki
JC
Ghosh
Gourisankar
G
Jennings
Patricia A
PA
Fu
Xiang-Dong
XD
Adams
Joseph A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
D009687:Nuclear Proteins; D010455:Peptides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000068103:Serine-Arginine Splicing Factors; C087728:SRPK1 protein, human; D017346:Protein Serine-Threonine Kinases