Signaling mechanisms that regulate leukocyte migration

The migration and recruitment of leukocytes during inflammation plays a key role in the development and progression of periodontal disease. The actin cytoskeleton plays an important role in the regulation of leukocyte migration and recruitment. OBJECTIVES: Investigate the signaling mechanisms that regulate leukocyte migration. ROCKII is a Serine/Threonine kinase that is an effector of Rho GTPases. The kinase activity of ROCKII is activated following RhoA binding to the central coiled-coil domain, thereby inhibiting the autoinhibitory activity of the C-terminus. However, the role of the ROCKII C- terminal PH domain in ROCK activity has not been fully investigated to date.

METHODS: Two separate pull-down experiments using the ROCKII-PH domain as bait were performed: one with a His-tagged ROCKII PH-domain and another with a control sample lacking recombinant protein were used in a pull-down experiment. Eluted proteins from both experiments were subjected to 2D SDS-PAGE followed by mass-spectrometry analysis. The Mascot search algorithm was used to match peptides derived from interacting partners with archived sequences to identify the unknown binding partners.

RESULTS: Database interrogation revealed interactions between ROCKII and a number of previously known partners like CRMP2 and profilin 2. Biochemical analyses confirm the interaction between ROCKII and these two proteins. We also identified the interaction between ROCKII and important vesicle trafficking proteins. These findings, led to the prediction that ROCKII localizes to membranous structures. To test this hypothesis, we have performed subcellular fractionation experiments using a sucrose step-gradient which enriches early endosomes in a known and characterized interphase between two distinct sucrose densities.

CONCLUSION: These experiments show that significant amounts of ROCKII are present in the early endosome fraction – an important trafficking organelle. Future studies will investigate the interaction between ROCKII and particular binding partners that are likely to be important for the regulation of leukocyte trafficking.