Investigation of protein-tyrosine phosphatases by in-gel assays.

Renaturation permits the detection of protein-tyrosine phosphatase (PTP) activities subsequent to separation by SDS-PAGE in the presence of the (32)P-labeled poly(Glu(4)Tyr) as a macromolecular substrate. An efficient corresponding method has been developed by Burridge and Nelson [Anal. Biochem. 232 (1995) 56]. We describe here the modification of the basic method, its extension to two-dimensional gel electrophoresis, and applications to identify PTPs in signaling complexes and reversibly oxidized PTPs. Particular attention is given to the preparation of samples, to interpretation of the results as well as to advantages and limitations of the technique. Immunodepletion and the use of cells from knockout animals have been successful in the identification of individual PTPs. Readily detectable in cell lysates are PTP-PEST, SHP-2, SHP-1, PTP1B, and T-cell PTP. A much greater complexity of activity bands is, to a large extent, due to the generation of active fragments of PTPs. In-gel detection of PTPs can be employed to monitor cell fractionations and potential PTP activity changes.

CD44: from adhesion molecules to signalling regulators.

Cell-adhesion molecules, once believed to function primarily in tethering cells to extracellular ligands, are now recognized as having broader functions in cellular signalling cascades. The CD44 transmembrane glycoprotein family adds new aspects to these roles by participating in signal-transduction processes--not only by establishing specific transmembrane complexes, but also by organizing signalling cascades through association with the actin cytoskeleton. CD44 and its associated partner proteins monitor changes in the extracellular matrix that influence cell growth, survival and differentiation.

UVA inactivates protein tyrosine phosphatases by calpain-mediated degradation.

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand-independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.