B1-binding partners p53, Smad4, rLHR, p27(kip1), cullin, and c-jun
B1-binding partners p53, Smad4, rLHR, p27(kip1), cullin, and c-jun and also a consensus CDK3 site Jab1-interacting sequence derived. We then determined that the consensus Jab1-interacting sequence was present at amino acid position 161 in LMP-1 (Table 2) and confirmed this by building of a mutant LMP-1, in which the residues NTED were mutated to AAAA inside the putative Jab1-interacting area. Binding on the wild-type and mutant proteins to Jab1 was tested in slot blot assays. In slot blot-binding assays performed with purified recombinant proteins, the biotinylated TAT MP-1 (wild-type) bound to both Smurf1 and Jab1 that had been immobilized onto nitrocellulose blots. Mutation with the Smurf1-interacting motif in LMP-1 (LMP-1Smurf1) resulted in loss of binding to Smurf1 without the need of affecting its binding affinity for Jab1. Similarly, mutation on the Jab1-interacting motif (LMP-1Jab1) resulted in loss of binding of LMP-1 to Jab1 with out affecting its interaction with Smurf1 (Fig. 6). As expected, the double mutant (Smurf1Jab1) lacking the necessary motifs for Smurf1 and Jab1 entirely failed to bind these target proteins. Within this experiment the specific activity from the biotin labeling was normalized by estimating the amount of biotins per protein molecule by means of 4-hydroxyazobenzene-2-carboxylic acid (HABA) assay kit (Pierce). This confirms that the LMP-1 mutants do not bind Smurf1/Jab1, as expected, and validates the use of mutantsMol Cell Biochem. Author manuscript; obtainable in PMC 2015 January 01.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptSangadala et al.Pageto comprehend the value of interaction of LMP-1 with Jab1 and Smurf1 in osteoblast differentiation.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptLMP-1 interactions with both Jab1 and Smurf1 are expected for complete LMP-1 activity We assessed the activity of purified recombinant LMP-1 and its mutant proteins in the BMP-reporter assay. The Jab- noninteracting LMP mutant (LMP1Jab1), like the Smurfnoninteracting LMP mutant (LMP-1Smurf1), ALDH1 Storage & Stability showed considerable loss of BMP-2potentiating activity as measured by relative luciferase activity (Fig. 7A). As expected, almost half of BMP-potentiating activity of LMP-1 was lost in each and every Smurf1 or Jab1 mutant. The double mutant (Smurf1Jab1) lacking the necessary motifs for Smurf1 and Jab1 exhibits complete loss of activity. This indicated that both Smurf1 and Jab1 interactions are vital for LMP-1 to completely potentiate BMP-2 activity. Alkaline phosphatase assay confirms each Smurf1 and Jab1 interactions are essential for complete LMP-1 activity and validates the BMP-reporter assay To confirm the physiologic relevance in the BMP-reporter assay, we measured alkaline phosphatase activity in response to BMP-2 (50 ng/mL) in C2C12 cells (Fig. 7B). Cells have been transduced with different types of TAT MP-1 for 24 h before remedy with BMP-2 for 3 days. Therapy with full length TAT MP-1 (wild-type) improved BMP-2 induction of alkaline phosphatase activity almost 4-fold when the TAT MP-1 mutants lacking either the Smurf1 or Jab1-interacting motifs showed only partial enhancement. As expected, the double mutant (Smurf1Jab1) lacking the essential motifs for Smurf1 and Jab1 absolutely fails to exhibit the potentiating activity on BMP-induced ALP activity. These findings using a extra physiologically relevant enzyme marker, closely mimicked the BMP-reporter assay outcomes observed above. Jab1 knockdown by siRNA causes elevatio.