Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle

Abstract

Skeletal muscles produce transient reactive oxygen species (ROS) in response to intense stimulation, disuse atrophy, heat stress, hypoxia, osmotic stress, stretch and cell receptor activation. The physiological significance is not well understood. Protein phosphatases (PPases) are known to be highly sensitive to oxidants and could contribute to many different signalling responses in muscle. We tested whether broad categories of PPases are inhibited by levels of acute oxidant exposure that do not result in loss of contractile function or gross oxidative stress. We also tested if this exposure results in elevated levels of global protein phosphorylation. Rat diaphragm muscles were treated with either 2,3-dimethoxy-1-naphthoquinone (DMNQ; 1, 10, 100 μm; a mitochondrial O 2 •− /H 2 O 2 generator) or exogenous H 2 O 2 (5, 50, 500 μm) for 30 min. Supernatants were assayed for serine/threonine PPase (Ser/Thr-PPase) or protein tyrosine PPase (PTP) activities. With the exception of 500 μm H 2 O 2 , no other oxidant exposures significantly elevated protein carbonyl formation, nor did they alter the magnitude of twitch force. DMNQ significantly decreased all categories of PPase activity at 10 and 100 μm and reduced PTP at 1 μm. Similar reductions in Ser/Thr-PPase activity were seen in response to 50 and 500 μm H 2 O 2 and PTP at 500 μm H 2 O 2 . ROS treatments resulted a dose-dependent increase in the phosphorylation states of many proteins. The data are consistent with the concept that PPases, within intact skeletal muscles, are highly sensitive to acute changes in ROS activity and that localized ROS play a critical role in lowering the barriers for effective phosphorylation events to occur in muscle cells, thus increasing the probability for cell signalling responses to proceed. Abbreviations DifMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DMNQ, 2,3-dimethoxy-1-naphthoquinone; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; PPase, phosphatase; PTP, protein tyrosine phosphatase; PPM, a class of serine/threonine phosphatases dependent on Mg 2+ /Mn 2+ ; PPP, phosphoprotein phosphatases, a class of serine/threonine phosphatases containing Fe 2+ /Zn 2+ ; PTP1B, an important protein tyrosine phosphatase in extracellular receptor kinase signalling; ROS, reactive oxygen species; Ser/Thr-PPase, serine/threonine phosphatase.

Trypanosome Lytic Factor-1 Initiates Oxidation-stimulated Osmotic Lysis of Trypanosoma brucei brucei

“…In bloodstream form T. brucei brucei, NEM caused depolarization of the plasma membrane potential, presumably through inhibition of H ϩ -ATPases (41). In addition to ATPases, phosphatases often contain redox-active cysteine residues that are sensitive to oxidation (42). In a related parasite, Trypanosoma rangeli, H 2 O 2 was shown to inhibit the activity of phosphatases in the outer membrane, and thiol-reducing agents reversed phosphatase inhibition (43).…”

Insulin elicits a ROS-activated and an IP3-dependent Ca2+ release; both impinge on GLUT4 translocation

“…We have previously shown that NOX2 generates ROS in skeletal muscle myotubes in response to insulin (Espinosa et al, 2009). A rise in cellular ROS promotes redox modifications of skeletal muscle proteins (Hidalgo et al, 2004) and a global 'phosphatase tone' (Wright et al, 2009) in both skeletal and cardiac muscle. The thiol (-SH) group of cysteine residues undergoes covalent reactions with oxidants, which produce modifications, such as S-glutathionylation, that give rise to functional alterations.…”

Oxidative Stress-Associated Protein Tyrosine Kinases and Phosphatases in Fanconi Anemia

“…Moreover, SHP-1 and SHP-2 activity could be inhibited by oxidative stress generated by ionizing radiation or low H 2 O 2 concentrations in a Ca 2 + -dependent NOS and an S-nitrosylation-dependent manner (9,66,72). Further investigation indicated that oxidative stress-induced inhibition on PTP activity is reversible (46,122,131). ROS accumulation resulting from cytokine treatment decreases the PTP1B activity by oxidative inactivation, which can be restored by the treatment with NAC, an ROS scavenger (46,57).…”

“…Excessive generation of ROS has been shown to activate multiple protein kinases, such as extracellular signal-regulated kinase (ERK)1/2, protein kinase B (PKB), and protein tyrosine kinases (PTKs) (25,31,43,50,118,125,127). Additionally, a variety of phosphatases are the sensitive targets of oxidative stress and may be inactivated under oxidizing conditions, which would amplify the effect of redox-linked activation of key protein kinases (60,110,111,131). Given that the balance between protein kinases and phosphatases dictates the overall phosphorylation state of cellular phosphoproteins, aberrant protein phosphorylation may exacerbate the pathophysiology of multiple human diseases and targeting them by small molecules therefore would be anticipated to ameliorate clinical symptoms (34,38,76,79).…”