REGULATION OF RHO KINASE LOCALIZATION BY ITS C TERMINUS
Document Type
Presentation
Start Date
22-10-2010 10:45 AM
End Date
22-10-2010 12:00 PM
Description
Rho kinase (ROCK) is a ser/thr kinase which is an important downstream effector of RhoA family of small GTPases. ROCK regulates cell migration, invasion, cytokinesis, apoptosis, and oncogenic transformation. The objective of this study was to understand the regulation of ROCK during cell migration. Traditionally Rho and ROCK have been thought to be important for the regulation of contractility at the rear end of a migrating cell. However we observed GFP tagged ROCK to be transiently localized at the leading edge of a migrating cell. Membrane localization could be mediated by two candidate domains, the RBD (Rho binding domain) or the PH (Pleckstrin Homology) domain. Studies with truncation mutants showed that both PH domain and RBD are necessary for ROCK localization. Next, we wanted to identify the stimulus for ROCK localization during cell migration. Cells transfected with GFP tagged ROCK were plated on collagen, which activates integrin engagement and polylysine (PLL), which is a positively charged surface mediating non specific attachment. We found that cells plated on collagen showed significantly greater membrane localization compared to those plated on PLL. To delineate the downstream localization signals activated by integrin engagement, Pl3 signaling was blocked using L Y290004, which is a pharmacological inhibitor of Pl3 kinase and RhoA pathway was blocked using C3 transferase, which is a cell permeable Rho inhibitor. Treatment with each of the inhibitors showed significant decrease in ROCK membrane localization. Our data indicate that the C terminus serves as a site of integration for two distinct localization signals that are activated upon integrin engagement. The next objective was to investigate the significance of ROCK localization at the leading edge. Studies using siRNA knockdown of ROCK II showed decrease in the number of retraction events and increase in persistence of protrusions. Thus ROCK regulates localized retraction at the leading edge. ROCK II knockdown cells also showed decreased focal adhesions and F actin as compared to control cells. Thus integrin engagement activates two distinct localization signals which are received at the C terminus of ROCK, which combine to target ROCK II at the leading edge and regulate membrane dynamics.
Recommended Citation
Kher, Swapnil; Rana, M.; and Worthylake, R., "REGULATION OF RHO KINASE LOCALIZATION BY ITS C TERMINUS" (2010). Dr. Joseph M. Moerschbaecher, III Graduate Research Day. 15.
https://digitalscholar.lsuhsc.edu/grad_rs/2010/poster1/15
REGULATION OF RHO KINASE LOCALIZATION BY ITS C TERMINUS
Rho kinase (ROCK) is a ser/thr kinase which is an important downstream effector of RhoA family of small GTPases. ROCK regulates cell migration, invasion, cytokinesis, apoptosis, and oncogenic transformation. The objective of this study was to understand the regulation of ROCK during cell migration. Traditionally Rho and ROCK have been thought to be important for the regulation of contractility at the rear end of a migrating cell. However we observed GFP tagged ROCK to be transiently localized at the leading edge of a migrating cell. Membrane localization could be mediated by two candidate domains, the RBD (Rho binding domain) or the PH (Pleckstrin Homology) domain. Studies with truncation mutants showed that both PH domain and RBD are necessary for ROCK localization. Next, we wanted to identify the stimulus for ROCK localization during cell migration. Cells transfected with GFP tagged ROCK were plated on collagen, which activates integrin engagement and polylysine (PLL), which is a positively charged surface mediating non specific attachment. We found that cells plated on collagen showed significantly greater membrane localization compared to those plated on PLL. To delineate the downstream localization signals activated by integrin engagement, Pl3 signaling was blocked using L Y290004, which is a pharmacological inhibitor of Pl3 kinase and RhoA pathway was blocked using C3 transferase, which is a cell permeable Rho inhibitor. Treatment with each of the inhibitors showed significant decrease in ROCK membrane localization. Our data indicate that the C terminus serves as a site of integration for two distinct localization signals that are activated upon integrin engagement. The next objective was to investigate the significance of ROCK localization at the leading edge. Studies using siRNA knockdown of ROCK II showed decrease in the number of retraction events and increase in persistence of protrusions. Thus ROCK regulates localized retraction at the leading edge. ROCK II knockdown cells also showed decreased focal adhesions and F actin as compared to control cells. Thus integrin engagement activates two distinct localization signals which are received at the C terminus of ROCK, which combine to target ROCK II at the leading edge and regulate membrane dynamics.