A Novel Signaling Perturbation and Ribozyme Gene Therapy Procedure to Block Rho-Kinase (ROK) Activation and Breast Tumor Metastasis
Abstract
Breast tumor cells (SP- 1) express a major cell adhesion molecule, CD44v 10, which binds the extracellular matrix component, hyaluronan (HA), at its external domain and interacts with various signaling molecules at its cytoplasmic domain. In this study we have determined that CD44v10 and Rho-Kinase (ROK) are physically associated as a complex in vivo. Using a recombinant fragment of ROK in particular, the pleckstrin homology (PH) domain and in vitro binding assays, we have detected a specific binding interaction between the PH domain of ROK and the cytoplasmic domain of CD44. Scatchard plot analysis indicates that there is a single high affinity CD44 binding site in the PH domain of ROK with an apparent dissociation constant (Kd) of 1 .76nM which is comparable to CD44 binding (Kd 1 .56nM) to intact ROK. These findings suggest that the PH domain is the primary ROK binding region for CD44. Furthermore, HA binding to SP- 1 cells promotes RhoA-mediated ROK activity which, in turn, increases phosphorylation of three different inositol 1, 4, 5-trisphosphate receptors (IP3Rs) IN PARTICULAR, SUBTYPE 1 (LP3Rl), and to a lesser extent subtype 2 (1P3R2) and subtype 3 (1P3R3) all known as 1P3-gated Ca2+ channels. The phosphorylated IP3Rl (but not 1P3R2 and 1P3R3) is enhanced in its binding to 1P3 which subsequently stimulates 1P3-mediated Ca2+ flux. Transfection of SP- 1 cells with ROK's PH cDNA significantly reduces ROK association with CD44v 10, and effectively inhibits ROK-mediated phosphorylation of IP3Rs and IP3R-mediated Ca2+ flux in vitro. The PH domain of ROK also functions as a dominant-negative mutant in vivo to block HA-dependent, CD44v10-specific intracellular Ca2+ mobilization and breast tumor cell migration. Taken together, we believe that CD44v 10 interaction with ROK plays a pivotal role in IP3R-mediated Ca2+ signaling during HA-mediated breast tumor cell migration.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2001
- Accession Number
- ADA403860
Entities
People
- Lilly Y. Bourguignon
Organizations
- University of Miami