The EGFR phosphatase RPTPγ is a redox-regulated suppressor of promigratory signaling.

Spatially organized reaction dynamics between proto-oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases determine EGFR phosphorylation dynamics in response to growth factors and thereby cellular behavior within developing tissues. We show that the reaction dynamics of mutual inhibition between RPTPγ phosphatase and autocatalytic ligandless EGFR phosphorylation enable highly sensitive promigratory EGFR signaling responses to subnanomolar EGF levels, when < 5% receptors are occupied by EGF. EGF thereby triggers an autocatalytic phospho-EGFR reaction by the initial production of small amounts of phospho-EGFR through transient, asymmetric EGF-EGFR2 dimers. Single cell RPTPγ oxidation imaging revealed that phospho-EGFR induces activation of NADPH oxidase, which in turn inhibits RPTPγ-mediated dephosphorylation of EGFR, tilting the autocatalytic RPTPγ/EGFR toggle switch reaction towards ligandless phosphorylated EGFR. Reversibility of this reaction to EGF is maintained by the constitutive phosphatase activity of endoplasmic reticulum-associated TCPTP. This RPTPγ/EGFR reaction at the plasma membrane causes promigratory signaling that is separated from proliferative signaling induced by accumulated, liganded, phosphorylated EGF-EGFR in endosomes. Accordingly, loss of RPTPγ results in constitutive promigratory signaling from phosphorylated EGFR monomers. RPTPγ is thus a suppressor of promigratory oncogenic but not of proliferative EGFR signaling.

Assessment of Protein Carbonylation and Protein Tyrosine Phosphatase (PTP) Oxidation in Vascular Smooth Muscle Cells (VSMCs) Using Immunoblotting Approaches.

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S-) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling.