Objectives of Research:
A highly investigated field for several decades now relates to insulin-dependent signal transmission networks. A key question having clinical significance as well is whether insulin effects can be manifested via alternative (insulin-independent) signaling pathways. Our long-term studies on the insulino-mimetic actions of vanadium [i.e. Karlish and Shechter, (1980) Nature 284, 556; Degani et al. (1981) Biochem-istry 20, 5795; Shechter and Ron (1986) J. Biol. Chem. 261, 14951; Shechter et al. (1992) Biochem-istry 30, 2063, reviewed in Shechter Y. (1990) Diabetes 39, 1] revealed that the metabolic effects of insulin can be fully manifested through insulin-receptor independent pathways. The identification and characterization of the 'key players' in this backup system is the main objectives of our research.
Vanadium salts that mimic virtually all the metabolic effects of insulin, have no or minor effect in activating the insulin-receptor tyrosine kinase (InsRTK) in intact cellular systems (reviewed in Shechter et al., 1995). This vanadate bypassing receptor activation was fully validated, using specific cell permeable blockers of InsRTK [Shisheva and Shechter (1992) Biochemistry 31, 2063]. Endogenous tyrosine-phosphorylation, however, is a prerequisite condition for manifesting the metabolic effects of insulin. We therefore searched for additional vanadium-activatable non-receptor protein tyrosine kinase. Such a protein has been identified in the cytosolic fraction of rat adipocytes (CytPTK, Shisheva and Shechter, 1993).
The relevant features of CytPTK, and the basic differences between CytPTK and the insulin-receptor are summarized in Table I.
CytPTK is activated several fold by vanadium salts and participates in several of the insulin-like effects manifested by vanadium salts.
Establishment of a cell-free system: The lack of a cell-free experimental system for studying insulin-dependent signaling pathways was the predominant factor in slowing down this field of research. Recently, however, we have managed to establish such a cell-free system for the investigation of vanadium-dependent mechanism(s) of action (Elberg et al., 1994). This experimental system allowed us to determine that CytPTK activation is preceded and dependent on inhibiting vanadium-sensitive protein phosphotyrosine phosphatases (PTPases, Elberg et al. , 1994; Elberg et al. , 1995, Li et al., 1995a, b). Most of the vanadate-sensitive PTPases are intrinsic plasma membrane proteins. The more relevant PTPases to manifest the bioresponses of insulin are now identified (in preparation). An additional non-receptor protein-tyrosine kinase that is activated by vanadate was identified as well (submitted). In summary, the key players of the backup system are PTPases, and nonreceptor protein tyrosine kinases. The 'cross-talk' relationships between these components is the main interest of our laboratory.
|estimated molecular-weight||53 kDa||350-400 kDa|
|requirement for bivalent anion||Co2+||Mn2+|
|good exogenous substrate||PolyGlu4Tyr||PolyGlu4Tyr|
|sensitivity to N-ethylmaleimide||no effect||inactivation|
|inhibition by staurosporine (IC50)||2 uM||8 uM|
Shisheva, A. and Shechter, Y. (1993) Role of cytosolic tyrosine kinase in mediating insulin-like actions of vanadate in rat adipocytes. J. Biol. Chem. 268: 6463-6469.
Shisheva, A. and Shechter, Y. (1993) Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes; dissociation from vanadate effect. Endocrinology 133: 1562-1568.
Elberg, G., Li, J. and Shechter, Y. (1994) Vanadium activates or inhibits receptor and non-receptor protein tyrosine kinases in cell-free experiments, dependent on its oxidation state. Possible role of endogenous vanadium in controlling cellular protein tyrosine kinase activity. J. Biol. Chem. 269: 9521-9527.
Li, J., Elberg, G., Gefel, D. and Shechter, Y. (1995a) Permolybdate and pertungstate-potent stimulators of insulin effects in rat adipocytes; mechanism of action. Biochemistry 34: 6216-6225.
Li, J., Elberg, G., Libman, J., Shanzer, A., Gefel, D. and Shechter, Y. (1995b). Insulin-like effects of tungstate and molybdate: mediation through insulin receptor independent pathways. Endocrine 3: 631-637.
Elberg, G., Li, J., Leibovitch, A. and Shechter, Y. (1995) Non-receptor cytosolic protein tyrosine kinases from various rat tissues. Biochim. Biophys. Acta 1296: 299-306.
Shechter, Y., Li, J., Elberg, G., Gefel, D., Meyerovitch, J. and Shisheva, A. (1995) J. Cell Biochem. 153: 39-47.
Shechter, Y., Li J., Meyerovitch J., Gefel D., Bruck R., Elberg G., Miller D.S., Shisheva A. (1995) Insulin-like actions of vanadate are mediated in an insulin-receptor-independent manner via non-receptor protein tyrosine kinases and protein phosphotyrosine phosphatases, Mol. and Cel. Biochem. 153: 39-47.
Goldwaser, I., Gefel, D., Gershonov, E., Fridkin, M. and Shechter, Y. (2000) Insulin-like effects of vanadium; basic and clinical implications. J. Inorganic Biochem. 80:21-25.
Sun, Q., Sekar, N., Goldwaseer, I., Gershonov, E., Fridkin, M. and Shechter, Y. (2000) Vanadate restores glucose-6-phosphate in diabetic rats; A mechanism to enhance glucose metabolism. Am. J. Physiol. Endocrinol. Metab. 279:E403-E410.
Gershonov, E., Goldwaser, I., Fridkin, M. and Shechter, Y. (2000) A novel approach for a water-soluble long-acting insulin; Design, preparation and analysis of [2-sulfo]-9-fluorenylmethoxycarbonyl-insulin (FMS3-insulin). J. Med. Chem. 43:2530-2537.
Goldwaser, Y., Qian, S., Gershonov, E., Fridkin, M. and Shechter, Y. (2000) Organic vanadium chelators potentiate vanadium-evoked glucose metabolism in vitro and in vivo. Establishing criteria for optimal chelators. Mol. Pharmacol. 58:738-746.
Yoram Shechter, Liana Preciado-Patt, Gideon Schreiber, and Mati Fridkin (2001) Prolonging the half-life of human interferon-a2 in circulation: Design, preparation, and analysis of (2-sulfo-9-fluorenylmthoxycarbonyl)7-interferon-a2. Proc. Natl. Acad. Sci. USA 98: 1212-1217.
Yoram Shechter, Itzhak Goldwaser, Iris Lavon, Eytan Gershonov, Brenda Mester, Marina Mironchik, Lianna Preciado-Patt and Mati Fridkin (2001) A new approach for prolonging the half-life of peptides, proteins and low-molecular-weight drugs in vivo. Drugs of the Future 26: 472-480.
Yoram Shechter, Itzhak Goldwaser and Mati Fridkin (2001) Historic perspective and recent developments on the insulin-like actions of vanadium: Toward developing vanadium based drugs for diabetes. J. Am. Chem. Soc., in press.