Processive phosphorylation of alternative splicing factor/splicing factor 2.

Area of Science:

• Molecular Biology
• Biochemistry
• Gene Regulation

Background:

• SR proteins are essential for pre-mRNA splicing, regulating constitutive and alternative splicing pathways.
• Phosphorylation of SR proteins by SR protein-specific kinases (SRPKs) controls their function.
• SRPKs exhibit unique specificity and efficiency in phosphorylating repetitive arginine/serine (RS) domains.

Purpose of the Study:

• To investigate the kinetic mechanism of human SRPK1 phosphorylating the model SR protein ASF/SF2.
• To elucidate how SRPK1 interacts with and modifies the RS domain of ASF/SF2.

Main Methods:

• Development of specialized kinetic experiments, including the start-trap strategy.
• Monitoring of ASF/SF2 phosphorylation progress curves in the presence and absence of an SRPK1 active site inhibitor peptide.
• Analysis of enzyme-substrate complex phosphate content over time to determine release rates.

Main Results:

• ASF/SF2 phosphorylation was unaffected when the inhibitor peptide was added concurrently with ATP.
• Pre-incubation with the inhibitor peptide revealed an exponential decline in phosphate content, indicating SRPK1 release rate.
• SRPK1 employs a fully processive catalytic mechanism, phosphorylating ASF/SF2's RS domain while remaining bound.

Conclusions:

• SRPK1 utilizes a processive mechanism for ASF/SF2 phosphorylation, keeping the substrate engaged during modification.
• This 'locked' mechanism ensures efficient and specific phosphorylation of the repetitive RS domains within ASF/SF2.
• Understanding SRPK1's catalytic mechanism provides insights into the regulation of pre-mRNA splicing.