These observations did agree well with phosphorylation enhancement results from dot blots

e docking interaction is critical for regulation of phosphorylation of ASF/SF2. The docking motif is generally rich in basic residues, conforming to a consensus sequence, and binds the acidic groove of SRPK1, located far from the active site. However, the identical sequences were not found in any RS repeat sequences of fish LB3. GST-pull down assay showed affinity binding of SRPK1with RS repeats. The binding strength is dependent on the length of RS repeats. Electrostatics where negatively charged surface interacts with positively charged RS motif seems to be enough to phosphorylate Ser-28. This finding is contrast to the results of previous studies that docking motif of ASF/SF2 plays an important role; the RS domain itself does not affect the mechanism of phosphorylation. The sequence of the acidic docking groove of SRPK1 is highly conserved among species. It suggests that SRPK1 distinguish substrates with docking motif from various substrates with short RS repeats only properly. SRPK1 docks near the C-terminus of the RS1 segment and phosphorylates the RS domain at multiple Ser residues, using directional and possessive mechanisms, described by the proposed grab-and-pull model. Our experimental results are consistent with this model. The SRPK1 strongly binds to the short stretch of the RS motif. Also consistent with our results is the recent finding that LBR has a short conserved peptide sequence, RSRSPGR, that overlaps the RS repeats and may serve as a docking motif for SRPK1. This indicates that long and short RS repeats may constitute the docking and/or affinity binding motif. Similarly, an RS motif was inserted to the best position for SRPK1 bound to initialize phosphorylation of PubMed ID: Ser-28 of gfLB3. The length of sequential RS repeats determined the strength of interaction between SRPK1 and its substrates and pulled far Ser residues into the active site, in a similar manner to ASF/SF2. The positioning and numbers of the inserted RS repeats upstream of the p34cdc2 target Ser of the individual fish LB3 appeared to be determined by evolution of the fish lineage. In general, investigation into the phosphorylation specificity of SR proteins has been very difficult owing to the repetitiveness of sequence. However, our study suggests that analysis of the phosphorylation of a single amino acid by using a pSer-specific antibody may help in the analysis of the unique phosphorylation mechanism of minor proteins with short RS/SR dipeptides repeats, as well as typical SR proteins with long RS repeats. Acknowledgements We are grateful to Y. Kato-Unoki for operation of DNA sequencing. We also thank for M. Hagiwara for kindly gift of SRPK inhibitor. The cost of publication was supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. This research has been supported in parts by JSPS KAKENHI Grant Number 19580207, 22580200 to A. Yamaguchi. Neutralization of VEGF-A with anti-VEGF-A therapies, such as bevacizumab or VEGF-A receptor inhibitors can result in pain, when given alone or in combination with chemotherapies. The MedChemExpress UNC0642 clinical findings that VEGF-A contributes to pain are supported by observations that inhibition of VEGF receptor 2 exacerbates peripheral neuronal damage, which is often associated with pain, and enhances pain behaviors in normal, nerve-injured and diabetic animals. 0969-9961/ 2014 University of Nottingham. Published by Elsevier Inc. This is