A Novel Signaling Perturbation and Ribozyme Gene Therapy Procedure to Block Rho-Kinase (ROK) Activation and Breast Tumor Metastasis
Abstract
Metastatic breast tumor Met-1 cells express CD44v3,8-10, a major adhesion receptor which binds extracellular matrix components at its extracellular domain and interacts with the cytoskeletal protein, ankyrin, at its cytoplasmic domain. In this study we have determined that CD44v3,8-10 and RhoA GTPases are physically associated in vivo, and that CD44v3,8-10-bound RhoA displays GTPase activity which can be inhibited by botulinum toxin C3-mediated ADP-ribosylation. in addition, we have identified a 160kDa Rho-Kinase (ROK) as one of the downstream targets for CD44v3,8-10-bound RhoA GTPase. Specifically, RhoA (complexed with CD44v3,8-10) stimulates ROK-mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v3,8-10. Most importantly, phosphorylation of CD44v3,8-10 by ROK enhances its interaction with the cytoskeletal protein, ankyrin. We have also constructed two ROK cDNA constructs which encode for proteins consisting of 537 amino acids DESIGNATED AS THE CONSTITUTIVELY ACTIVE FORM OF ROK containing the catalytic domain (CAT, also the kinase domain), and 173 amino acids DESIGNATED AS THE DOMINANT-NEGATIVE FORM OF ROK containing the Rho-binding domain (RB). Microinjection of the ROK's CAT domain into Met-1 cells promotes CD44-ankyrin associated membrane ruffling and projections.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2000
- Accession Number
- ADA393016
Entities
People
- Lilly Y. Bourguignon
Organizations
- University of Miami