Kinase Domain Insertions Define Distinct Roles of CLK Kinases in SR Protein Phosphorylation

Alex N Bullock,Ann L Amos,Evangelos Papagrigoriou,Peter Rellos,Judit É Debreczeni,Gourisankar Ghosh,Sanjan Das,Frank H Niesen,Kunde Guo,Suhyung Cho,Benjamin E Turk,Stefan Knapp,Oleg Fedorov

doi:10.1016/j.str.2008.12.023

Alex N Bullock, Ann L Amos + Show 11 more

Open Access

https://doi.org/10.1016/j.str.2008.12.023

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Abstract

SummarySplicing 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 αH. In addition, substrate docking grooves present in related mitogen activating protein kinases (MAPKs) are inaccessible due to a CLK specific β7/8-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.

Highlights

Cdc2-like kinases (CLK) are conserved throughout eukaryote evolution, from Saccharomyces cerevisiae (KNS1), Arabidopsis thaliana (AFC1-3), and Drosophila melanogaster (DOA) to mouse (CLK/STY) and human (CLK1-4) (Yun et al, 1994)
In humans, splicing of the homolog hTRA2beta is regulated by CLK2 (Stoilov et al, 2004) whereas CLK1 plays an important role in neuronal differentiation (Myers et al, 1994)
Overview of the CLK1 and CLK3 Structures The kinase domains of CLK1 and CLK3 were expressed in E. coli in active form as demonstrated by extensive autophosphorylation

Summary

Introduction

Cdc2-like kinases (CLK) are conserved throughout eukaryote evolution, from Saccharomyces cerevisiae (KNS1), Arabidopsis thaliana (AFC1-3), and Drosophila melanogaster (DOA) to mouse (CLK/STY) and human (CLK1-4) (Yun et al, 1994). Based on an unique and conserved ‘‘EHLAMMERILG’’ signature motif, CLKs are often referred to as ‘‘LAMMER kinases.’’ The conserved signature motif of CLKs has been suggested to dictate CLK substrate specificity (Yun et al, 1994). Consequences of CLK dysfunction are dramatically illustrated in Drosophila by defects in the CLK homolog DOA (darkener of apricot). The doa gene has an essential role in Drosophila embryogenesis; it is expressed throughout development and its mutation leads to defects in segmentation, eye formation, and neuronal development (Yun et al, 1994). In humans, splicing of the homolog hTRA2beta is regulated by CLK2 (Stoilov et al, 2004) whereas CLK1 plays an important role in neuronal differentiation (Myers et al, 1994). CLK3 is abundantly expressed in mature spermatozoa and might play a role in the fertilization process (Menegay et al, 1999)

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