A Novel Signaling Perturbation and Ribozyme Gene Therapy Procedures to Block Rho-Kinase (ROK) Activations and Breast Tumor Metastasis
Abstract
In breast tumor cells (e.g. Met-i and SP-t cell lines), the Rho-Kinase (ROK) is detected as a l6OkDa protein. We have demonstrated that ROK phosphorylates the cytoplasmic domain of CD44 A HYALURONAN (HA) receptor and up-regulates the interaction between CD44 and the cytoskeletal protein, ankyrin during HA/CD44-regulated breast tumor cell migration. Most recently, we have found that CD44 and Rho-Kinase (ROK) are also physically associated as a complex in breast tumor cells. Biochemical analyses show that the C-terminal pleckstrin homology (PH) domain is the primary ROK binding region for CD44. Most importantly, HA binding to' cells promotes RhoA-mediated ROK activity which, in turn, increases phosphorylation of three different inositol 1, 4, 5-trisphosphate receptors (IP3Rs) IN PARTICULAR, SUBTYPE I (IP3Rl), 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 or 1P3R3) is enhanced in its binding to 1P3 which subsequently stimulates 1P3-mediated Ca2+ flux and breast tumor cell migration. We have also constructed two dominant-negative ROK cDNA constructs which encode for the Rho- binding (RB) domain and the pleckstrin homology (PH) domain. Our data indicate that transfection of breast tumor cells with ROK's RB or PHcDNA significantly blocks HA and CD44-induced Ca2+ signaling and breast tumor cell migration. Taken together, we believe that ROK plays a pivotal role in CD44-cytoskeleton interaction and IP3R-mediated Ca2+ signaling during HA-mediated breast tumor progression.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2002
- Accession Number
- ADA410099
Entities
People
- Lily Y. Bourguignon
Organizations
- Northern California Institute for Research and Education