PH Domain Of CKIP-1Interacts With Ubiuitin

CKIP-1(casein kinase2interacting protein-1) was originally identified as a specificCK2ɑ-interacting protein. The CKIP-1protein contains a plecktrin homology (PH)domain at the N-terminus and a putative leucine zipper (LZ) motif at the C-terminus,which could mediate multiple interactions between CKIP-1and numerous cellularproteins. Based on the findings at the molecular and cellular levels, CKIP-1is involvedin various important signaling pathways, such as controlling cell growth, apoptosis,differentiation, cytoskeleton and plasma membrane recruitment of ATM and CK2.Research on in vivo physiological role of CKIP-1has revealed it functions as anenhancement factor of Smurf1. Ubiquitin-proteasome pathway plays crucial roles incellular physiological function through selective removal of misfolded and specificproteins. Protein ubiquitination process is catalyzed sequentially byubiquitin-activating enzyme E1, ubiquitin conjugating enzyme E2and ubiquitinprotein ligase E3to label substrate proteins with ubiquitin chains which promote theirdegradation in the26S proteasome. Smurf1(Smad ubiquitination regulatory factor1)was originally identified as an E3ligase of TGF-β/BMP pathway signaling moleculesSmad1/5. RhoA, Runx2and phosphorylated form of MEKK2were subsequently foundto be other substrates of Smurf1. Smurf1plays a negative role in bone formation.We have previously found that C-terminal of CKIP-1can interact with linker betweenWW domains of Smurf1and enhance the ligase activity of Smurf1. Mice deficient ofCKIP-1display increased bone mass and augmented osteoblast activity, phenotypeswhich are similar to that of Smurf1knockout mice. Meanwhile, we have also found that CKIP-1can also interact directly with the Rpt6ATPase of the19S regulatoryparticle of the proteasome through its leucine zipper. CKIP-1mediates theSmurf1-Rpt6interaction and delivers the ubiquitylated substrates to the proteasome.Thus, CKIP-1plays dual functions in regulation of Smurf1by inducingSmurf1-substrate interaction or by recuiting them to the26S proteosome via Rpt6subunit. Recently, we together with Nankai University resolved the crystal structure ofPH domain of CKIP-1and single crystals diffracted to1.7A resolution. Paperpublished on Nature at2008reported Rpn13functions as an ubiquitin receptor,attracted our attentions. The structure of Rpn13is determined and found to contain apleckstrin-homology domain (PHD) fold. The co-complex of Rpn13and ubiquitinreveals that the loops of PHD other than secondary structural elements in Rpn13areused to capture ubiquitin. So we wonder whether PH domain of CKIP-1can interactwith ubiqitin.In the present work, we used homology modeling, molecular docking and GST pulldown assay to examine the interaction between PH domain of CKIP-1and ubiquitin.Crystal structure of PH domain of CKIP-1showed three deletion regions (60,63-65and82-91) at the loops. So we first optimized the structure of PH domain of CKIP-1by homology modeling. Then, we predicted the binding models of PH domain ofCKIP-1with ubiquitin and found the key residues by molecular docking.4optimalinteracting models of PH domain and ubiquitin were screened and16key residueswere chosen for further biochemical analysis. Finally, we tested the binding ability ofPH domain and PH-mutant with ubiquitin by GST pull-down assay. Our resultsdemonstrated that PH domain of CKIP-1could interact with ubiquitin and mutations ofkey residues in PH domain could reduce the binding ability. Based on our research, wespeculate that PH domain of CKIP-1may promote the ubiquitin transfer to Smurf1andthen increase the ubiquitin ligase activity of Smurf1. The current findings will help to further reveal the mechanism of CKIP-1PH domainin Smurf1-ubiquitin-proteasome pathway. Both of N-terminal PH domain andC-terminal LZ domain of CKIP-1may be potential drug target for bone anabolicstrategy.