Allosteric Interactions Direct Binding and Phosphorylation of ASF/SF2 by SRPK1

Huynh, Nhat; Ma, Chen‐Ting; Giang, Ngoc; Hagopian, Jonathan C.; Ngo, Jacky Chi Ki; Adams, Joseph A.; Ghosh, Gourisankar

doi:10.1021/bi901107q

Cited by 16 publications

(14 citation statements)

References 17 publications

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“…While removal of the RRMs in ASF/SF2 enhances PP1 activity toward the RS domain by about an order of magnitude (Fig. 6B), removal of these domains does not enhance SRPK1 phosphorylation activity 25. Crystallographic and deletion analyses show that the sufficiency of the RS domain for SRPK1 phosphorylation is due to a well-contoured, extensive binding pocket that tightly recognizes the RS1 segment 21…”

Section: Discussionmentioning

confidence: 98%

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Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

Ghosh

et al. 2010

Journal of Molecular Biology

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SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, region-specific mapping of phosphorylation sites in ASF/SF2 was monitored as a function of the protein phosphatase PP1. We showed that ten phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the Cto-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs (RRMs) in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RRMs protects against dephosphorylation suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.Keywords protein kinase; protein phosphatase; phosphorylation; splicing; SR protein Long precursor mRNA is converted to shorter mRNA strands for protein translation at a macromolecular complex (spliceosome) composed of several snRNPs (U1, U2, U4, U5, & U6) and more than one hundred auxilliary proteins. 1 Among the latter group, the SR

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Section: Discussionmentioning

confidence: 98%

“…All His-tagged ASF/SF2 constructs were refolded and purified using a published protocol 17. GST-tagged ASF/SF2 cleavage construct was purified by glutathione-resin affinity chromatography using a published procedure 25. SRPK1 and Clk/Sty were purified by Ni-resin affinity chromatography using a published procedure 18…”

Section: Methodsmentioning

confidence: 99%

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

Ghosh

et al. 2010

Journal of Molecular Biology

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“…SRSF1, SRSF2, SRSF5, and TRA2β1 were refolded and purified as previously published [15]. GST protein constructs were purified with glutathione-agarose resin according to a published procedure [20]. …”

Section: Methodsmentioning

confidence: 99%

N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

Aubol

Plocinik

Keshwani

et al. 2014

Biochemical Journal

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SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine-serine-rich) domains by two major families of protein kinases. The SRPKs efficiently phosphorylate the arginine-serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs lack a docking groove and phosphorylate both arginine-serine and serine-proline dipeptides, modifications that generate a hyper-phosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long, flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1. This interaction not only is essential for facilitating hyper-phosphorylation but also induces cooperative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2β1 by 2–3-fold. These findings suggest that CLK1-dependent hyper-phosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

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“…The docking groove initially binds the N-terminal portion of RS1 feeding the C-terminal end into the active site for phosphorylation initiation. The N-terminal Arg-Ser repeats then sequentially translate from the docking groove to the active site, a process that leads to unfolding of the final β strand in RRM2 which then occupies the docking groove (27). Rapid quench flow studies indicate that the rate of serine phosphorylation and translocation of the Arg-Ser stretch through the docking groove and active site are fast whereas the release of the product ADP is rate-limiting at each step (11).…”

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confidence: 99%

Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

et al. 2012

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Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases. However, the kinase domains of protein kinases are commonly flanked by sequences that regulate catalytic function. To address whether such sequences could alter nucleotide exchange and, thus, regulate protein phosphorylation, the presence of activating residues external to the kinase domain was probed in the serine protein kinase SRPK1. Deletion analyses indicate that a small segment of a large spacer insert domain and a portion of an N-terminal extension function cooperatively to increase nucleotide exchange. The data point to a new mode of protein kinase regulation in which select sequences outside the kinase domain constitute a nucleotide release factor that likely interacts with the small lobe of the kinase domain and enhances protein substrate phosphorylation through increases in ADP dissociation rate.

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Allosteric Interactions Direct Binding and Phosphorylation of ASF/SF2 by SRPK1

Cited by 16 publications

References 17 publications

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

Nucleotide Release Sequences in the Protein Kinase SRPK1 Accelerate Substrate Phosphorylation

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