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Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation.

Bullock AN, Das S, Debreczeni JE, Rellos P, Fedorov O, Niesen FH, Guo K, Papagrigoriou E, Amos AL, Cho S, Turk BE, Ghosh G, Knapp S - Structure (2009)

Bottom Line: In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific beta7/8-hairpin insert.Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2.In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK. alex.bullock@sgc.ox.ac.uk

ABSTRACT
Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix alphaH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific beta7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.

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Inhibitor Binding and Specificity(A) Binding of debromohymenialdisine to CLK1. Electron density (2FoFc) for the inhibitor is shown in blue.(B) Chart showing CLK1 and CLK3 kinase activities relative to the reference in the presence of 100 nM of 11 compounds causing the highest Tm shifts.(C) Table of the inhibitor data with corresponding chemical structures.
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fig7: Inhibitor Binding and Specificity(A) Binding of debromohymenialdisine to CLK1. Electron density (2FoFc) for the inhibitor is shown in blue.(B) Chart showing CLK1 and CLK3 kinase activities relative to the reference in the presence of 100 nM of 11 compounds causing the highest Tm shifts.(C) Table of the inhibitor data with corresponding chemical structures.

Mentions: CLK1 was crystallized in the presence of hymenialdisine, a marine sponge metabolite that has nanomolar activity against CDKs, MEK1, GSK3β, and CK1 (Meijer et al., 2000). In the crystal structure, hymenialdisine is bound in the typical ATP-mimetic manner, at the hinge backbone region where residues Glu242, Leu243, and Leu244 interact through one water-mediated and two direct main-chain hydrogen bonds. In addition, further hydrogen bonds are observed with conserved residues Asp305, Lys191, and Asn294 resembling the well-conserved binding mode of this inhibitor class (Figure 7A). No density was observed for the bromide substituent, suggesting that the halogen bond was hydrolyzed to yield debromohymenialdisine.


Kinase domain insertions define distinct roles of CLK kinases in SR protein phosphorylation.

Bullock AN, Das S, Debreczeni JE, Rellos P, Fedorov O, Niesen FH, Guo K, Papagrigoriou E, Amos AL, Cho S, Turk BE, Ghosh G, Knapp S - Structure (2009)

Inhibitor Binding and Specificity(A) Binding of debromohymenialdisine to CLK1. Electron density (2FoFc) for the inhibitor is shown in blue.(B) Chart showing CLK1 and CLK3 kinase activities relative to the reference in the presence of 100 nM of 11 compounds causing the highest Tm shifts.(C) Table of the inhibitor data with corresponding chemical structures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2667211&req=5

fig7: Inhibitor Binding and Specificity(A) Binding of debromohymenialdisine to CLK1. Electron density (2FoFc) for the inhibitor is shown in blue.(B) Chart showing CLK1 and CLK3 kinase activities relative to the reference in the presence of 100 nM of 11 compounds causing the highest Tm shifts.(C) Table of the inhibitor data with corresponding chemical structures.
Mentions: CLK1 was crystallized in the presence of hymenialdisine, a marine sponge metabolite that has nanomolar activity against CDKs, MEK1, GSK3β, and CK1 (Meijer et al., 2000). In the crystal structure, hymenialdisine is bound in the typical ATP-mimetic manner, at the hinge backbone region where residues Glu242, Leu243, and Leu244 interact through one water-mediated and two direct main-chain hydrogen bonds. In addition, further hydrogen bonds are observed with conserved residues Asp305, Lys191, and Asn294 resembling the well-conserved binding mode of this inhibitor class (Figure 7A). No density was observed for the bromide substituent, suggesting that the halogen bond was hydrolyzed to yield debromohymenialdisine.

Bottom Line: In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific beta7/8-hairpin insert.Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2.In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.

View Article: PubMed Central - PubMed

Affiliation: Structural Genomics Consortium, University of Oxford, Old Road Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK. alex.bullock@sgc.ox.ac.uk

ABSTRACT
Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences between splicing factor targeting kinases, we determined crystal structures of CLK1 and CLK3. Intriguingly, in CLKs the SRPK1 docking site is blocked by insertion of a previously unseen helix alphaH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific beta7/8-hairpin insert. Thus, the unconstrained substrate interaction together with the determined active-site mediated substrate specificity allows CLKs to complete the functionally important hyperphosphorylation of splicing factors like ASF/SF2. In addition, despite high sequence conservation, we identified inhibitors with surprising isoform specificity for CLK1 over CLK3.

Show MeSH
Related in: MedlinePlus