Dept. of Neuroscience
Oregon Health & Science University
In hair cells of the inner ear, the process of adaptation resets the open probability of the transduction channel to its resting value restoring hair-cell sensitivity. Determining how the adaptation motor, myosin-1c (Myolc), is regulated is critical for understanding how sensitivity is controlled in hair cells. Previous research has shown that activators of Protein Kinase A (PKA) can reduce channel open probability, suggesting a decrease in force production by the adaptation motor. Consistent with these results, a PKA phosphorylation site (RRXS) is located at serine 701 (S701) in a hinge region between the head and neck of Myolc, a prime location to affect mechanics of the motor. The experiments herein demonstrate that Myolc could be phosphorylated by PKA at S701. Short peptides of Myolc containing the PKA consensus site at S701 were strongly phosphorylated by PKA. Sf9 cell-expressed constructs of Myolc containing either the head or tail domains in addition to the neck domains were also phosphorylated by PKA, though stoichiometry measurements showed only 10-15% of the protein was phosphorylated. Phosphorylation levels of long constructs were increased in the presence of Ca[superscript 2+]. Phosphorylation of short peptides containing only the first IQ domain and PKA consensus site were phosphorylated by PKA strongly in the presence of calmodulin (CaM), but inhibited when Ca[superscript 2+] was present. CaM also bound with equal affinity to the first IQ domain of both phosphorylated and unphosphorylated short peptides of xiv Myolc. These results suggest that the absence of CaM from either the second, third, or fourth IQ domain may be ideal for phosphorylation. In an attempt to determine the phosphorylation state of Myolc in hair cells, the Myolc phosphorylation states in COS-7 cells and purified Myolc from Sf9 cells were measured. Cells were exposed to reagents to stimulate or inhibit phosphorylation, or no treatment, and then immunoprecipitated with an antibody specific for phosphorylated Myolc. When immunoprecipitated eluates were probed with a general Myolc antibody, no Myolc was recognized. These results show that Myolc is unphosphorylated both in COS-7 and Sf9 cells. These results confirm that Myolc is difficult to phosphorylate, or may require other undetermined conditions for efficient phosphorylation. To determine the importance of the S701 site for Myolc function, the ATPase and in vitro motility activities were was investigated in head-neck (HN) Myolc mutants that mimic the phosphorylated (HNS701D) and unphosphorylated (HNS701A) states of Myolc. Preliminary results show that HNS701D had slower ATPase rates than HNS701A or HN. Both HN701D and HNS701A translocated actin at slower rates than HN, with HN701D displaying the slowest velocities. In an assay of force production, the actin-binding protein Î±-actinin was introduced into the in vitro motility experimental set-up. Predictably, HN was stalled with increasing concentrations Î±-actinin. The velocities of both HN701D and HN701A remained constant with increasing amounts of Î±-actinin. These results suggest that both HN701A and HN701D have greater force-producing capabilities than xv HN. These results also illustrate that importance of the S701 site for Myolc mechanics. Finally, in vitro motility studies showed that wild-type Myolc had a greater velocity than the truncated version HN. These results suggest the tail region is potentially critical for the myosin step size or force production and may play a role in stabilization or unfolding of the protein to effectively translocate actin. Myolc was also determined to be a key component in the process of fast adaptation. To support in vivo studies in mice, in vitro motility studies were conducted with the in vitro motility assay. NMB-ADP, the mutant-specific inhibitor of Y61G Myolc, effectively slowed down motor velocity and had no effect on wild type mouse Myolc, as expected. Ca[superscript 2+] was also able to inhibit velocity of both wild type and Y61G Myolc. These results illustrate the complicated nature of phosphorylating Myolc. We have determined that the presence of CaM at IQ1 and Ca[superscript 2+] is crucial for phosphorylation of both short and long constructs of Myolc. These results suggest that CaM at IQ2 or an intermediate Ca[superscript 2+] occupied state of CaM may influence PKA phosphorylation of S701. Mutations of S701 also affected ATPase rate, in vitro motility velocities, and force-producing properties of Myolc confirming that S701 is critical for Myolc mechanics. Taken together, these results support the preliminary effects of PKA inhibitors and activators on hair cell open probability.
School of Medicine
Miller, Emilie Diane, "Phosphorylation of myosin-1c by cAMP-dependent protein kinase a dissertation" (2007). Scholar Archive. 184.