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  current news   Press   selected story    
     
  30th September 2010  
 

The direct association of SPRED1 or SPRED2 with DYRK1A modifies substrate/kinase interactions.

 
 




Authors:
Dan Li, Rebecca A. Jackson, Permeen Yusoff and Graeme R. Guy

Signal Transduction Laboratory, IMCB, Proteos, Singapore.

Published in Journal of Biological Chemistry, Aug 24, 2010. [Epub ahead of print].

Abstract
The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins comprise a family of three members: SPRED1-3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in C. elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down Syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates: Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A.

 
 


 
 


Figure Legend: SPRED1, SPRED2 and Spry2 disrupt the DYRK1A directed substrate phosphorylation.
A. Cell lysates from 293 cells transfected with the indicated plasmids (WT-DYRK1A, KD-DYRK1A and vector control) were subjected to SDS-PAGE and immnoblotted with anti-phospho-Tau Thr212 (p-Tau Thr212), anti-Tau and anti-HA. B. Cell lysates from 293 cells transfected with increasing amounts of DYRK1A and a constant amount of Tau were subjected to SDS-PAGE and immnoblotted with anti-phospho-Tau (p-Tau Thr212), anti-Tau and anti-HA. C. 293 cells were transfected with constant amount of WT-DYRK1A and Tau. The cells were treated with increasing concentrations of harmine for 30 min after 24 h post-transfection. The cell lysates were subjected to SDS-PAGE and immnoblotted with the antibodies indicated on the left. D. Cell lysates from 293 cells co-transfected with WT-, KD-DYRK1A, Spry2 and Tau were subjected to SDS-PAGE and immnoblotted with the indicated antibodies. E. Cell lysates from 293 cells transfected with combinations of WT-, KD-DYRK1A, SPRED1, SPRED2 and Tau were subjected to SDS-PAGE and immunoblotted with the antibodies shown on the left. The relative quantities of phospho-Tau for each lane in Fig. 5B-5E (top panel) were indicated in the bar charts (Supplemental Figure 5 A-D, respectively). F. Cell lysates from 293 cells co-transfected with WT-, KD-DYRK1A, SPRED1, SPRED2 and STAT3 were subjected to SDS-PAGE and immnoblotted with the indicated antibodies. The relative levels of phospho-STAT3 are indicated in the bar chart (Supplemental Figure 5E).

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