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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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CLK β-Hairpin Insert(A) Overview showing the location of the β-hairpin insert conserved within the CLK family. The hairpin interacts with a groove formed by helix αD and αE. Shown is the CLK1 β-hairpin (βhp and βhp′) in yellow and the CLK3 β-hairpin in blue. A docking peptide of the MAPK p38 (PDB code 1LEW) is shown in ball-and-stick representation (Chang et al., 2002). A detailed view of the hydrophobic interaction formed by Leu316 conserved within the CLK family (CLK3 is shown in blue). Leu316 superimposes well with a leucine present at this position in the MEF2A-derived MAPK docking peptide (colored in white). A Sequence alignment of human CLK family β-hairpin inserts is shown under the peptide interaction insert. Conserved residues are highlighted in red, the conserved hydrophobic residues making contact with the binding groove are highlighted in yellow and marked by an asterisk. The disordered tip of the hairpin insert is shown by a dotted yellow line.(B) Interactions formed by CLK1 β-hairpin residues with the surface of CLK1. Residues present in the hairpin are labeled in dark blue and those present in the kinase lower lobe are labeled in red.
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fig3: CLK β-Hairpin Insert(A) Overview showing the location of the β-hairpin insert conserved within the CLK family. The hairpin interacts with a groove formed by helix αD and αE. Shown is the CLK1 β-hairpin (βhp and βhp′) in yellow and the CLK3 β-hairpin in blue. A docking peptide of the MAPK p38 (PDB code 1LEW) is shown in ball-and-stick representation (Chang et al., 2002). A detailed view of the hydrophobic interaction formed by Leu316 conserved within the CLK family (CLK3 is shown in blue). Leu316 superimposes well with a leucine present at this position in the MEF2A-derived MAPK docking peptide (colored in white). A Sequence alignment of human CLK family β-hairpin inserts is shown under the peptide interaction insert. Conserved residues are highlighted in red, the conserved hydrophobic residues making contact with the binding groove are highlighted in yellow and marked by an asterisk. The disordered tip of the hairpin insert is shown by a dotted yellow line.(B) Interactions formed by CLK1 β-hairpin residues with the surface of CLK1. Residues present in the hairpin are labeled in dark blue and those present in the kinase lower lobe are labeled in red.

Mentions: The most striking feature of the CLK C-terminal lobe is a long insertion between the two sheets β7 and β8. This insert forms the CLK-specific βhp-βhp′ hairpin that folds over a shallow groove created by the helices αD and αE (Figure 3). Interestingly, substrates of MAPKs recognize the same shallow groove through so-called D motif docking sites by using mainly hydrophobic contacts. Although MAPK docking peptides bind in an orientation that is rotated approximately 90° compared with the β-hairpin, their central hydrophobic patch is mimicked by a conserved hydrophobic residue in the β-hairpin (Figures 3A and 3B). This β-hairpin structure is common to CLK1 and CLK3. Although there is low sequence homology in the region, a number of positions in the β-hairpin are conserved throughout the CLK family (Figure 3C). Apart from the hydrophobic patch (Leu316 in CLK1) and a conserved valine (Val297), the β-hairpin is docked to the shallow groove by a number of hydrogen bonds, including backbone contacts formed by the side chains of Arg311 and Ser299 in CLK1. This hairpin is also the site of a structurally uncharacterized insertion of ∼250 residues in SRPK1.


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)

CLK β-Hairpin Insert(A) Overview showing the location of the β-hairpin insert conserved within the CLK family. The hairpin interacts with a groove formed by helix αD and αE. Shown is the CLK1 β-hairpin (βhp and βhp′) in yellow and the CLK3 β-hairpin in blue. A docking peptide of the MAPK p38 (PDB code 1LEW) is shown in ball-and-stick representation (Chang et al., 2002). A detailed view of the hydrophobic interaction formed by Leu316 conserved within the CLK family (CLK3 is shown in blue). Leu316 superimposes well with a leucine present at this position in the MEF2A-derived MAPK docking peptide (colored in white). A Sequence alignment of human CLK family β-hairpin inserts is shown under the peptide interaction insert. Conserved residues are highlighted in red, the conserved hydrophobic residues making contact with the binding groove are highlighted in yellow and marked by an asterisk. The disordered tip of the hairpin insert is shown by a dotted yellow line.(B) Interactions formed by CLK1 β-hairpin residues with the surface of CLK1. Residues present in the hairpin are labeled in dark blue and those present in the kinase lower lobe are labeled in red.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: CLK β-Hairpin Insert(A) Overview showing the location of the β-hairpin insert conserved within the CLK family. The hairpin interacts with a groove formed by helix αD and αE. Shown is the CLK1 β-hairpin (βhp and βhp′) in yellow and the CLK3 β-hairpin in blue. A docking peptide of the MAPK p38 (PDB code 1LEW) is shown in ball-and-stick representation (Chang et al., 2002). A detailed view of the hydrophobic interaction formed by Leu316 conserved within the CLK family (CLK3 is shown in blue). Leu316 superimposes well with a leucine present at this position in the MEF2A-derived MAPK docking peptide (colored in white). A Sequence alignment of human CLK family β-hairpin inserts is shown under the peptide interaction insert. Conserved residues are highlighted in red, the conserved hydrophobic residues making contact with the binding groove are highlighted in yellow and marked by an asterisk. The disordered tip of the hairpin insert is shown by a dotted yellow line.(B) Interactions formed by CLK1 β-hairpin residues with the surface of CLK1. Residues present in the hairpin are labeled in dark blue and those present in the kinase lower lobe are labeled in red.
Mentions: The most striking feature of the CLK C-terminal lobe is a long insertion between the two sheets β7 and β8. This insert forms the CLK-specific βhp-βhp′ hairpin that folds over a shallow groove created by the helices αD and αE (Figure 3). Interestingly, substrates of MAPKs recognize the same shallow groove through so-called D motif docking sites by using mainly hydrophobic contacts. Although MAPK docking peptides bind in an orientation that is rotated approximately 90° compared with the β-hairpin, their central hydrophobic patch is mimicked by a conserved hydrophobic residue in the β-hairpin (Figures 3A and 3B). This β-hairpin structure is common to CLK1 and CLK3. Although there is low sequence homology in the region, a number of positions in the β-hairpin are conserved throughout the CLK family (Figure 3C). Apart from the hydrophobic patch (Leu316 in CLK1) and a conserved valine (Val297), the β-hairpin is docked to the shallow groove by a number of hydrogen bonds, including backbone contacts formed by the side chains of Arg311 and Ser299 in CLK1. This hairpin is also the site of a structurally uncharacterized insertion of ∼250 residues in SRPK1.

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