Intención para involucrarse en la puesta en valor los recursos

Alexander James Hale

_{, Jelmer Hoeksma}

_,

and Jeroen den Hertog

1. Hubrecht Institute – KNAW and University Medical Center Utrecht,

Utrecht, the Netherlands

7

AbstRACt

The production of reactive oxygen species (ROS) directly following amputation of the zebrafish embryo caudal fin-fold is essential for proper caudal fin-fold regeneration. Protein-tyrosine phosphatases (PTPs) are enzymes that dephosphorylate phospho- tyrosine through a mechanism involving a catalytic cysteine, and are susceptible to reversible inactivation by ROS. Although Src homology 2 domain containing phospha-

tase Shp2a is oxidized following zebrafish caudal fin amputation, the catalytic activity of Shp2a is required for caudal fin-fold regeneration. Here, we show that mutation of

the back-door cysteines C334 and C368 of Shp2a, which are proposed to protect the

catalytic cysteine from irreversible oxidation, reduced the catalytic activity of Shp2a and enhanced the susceptibility of Shp2a to oxidation. Surprisingly, expression of

these back-door cysteine mutants in ptpn11a-/-_ptpn11b-/- _{zebrafish embryos, which}

lack functional Shp2, rescued caudal fin-fold regeneration. As the back-door cysteine mutants of Shp2a demonstrated reduced catalytic activity, we investigated the level of Shp2a catalytic activity required for its function in zebrafish embryo caudal fin-fold regeneration by using two mutants of Shp2a, Shp2a-D61G and Shp2a-A462T, which have increased and reduced catalytic activity respectively. Interestingly, expression of Shp2a-D61G or Shp2a-A462T in ptpn11a-/-_ptpn11b-/- _{zebrafish embryos rescued caudal}

fin-fold regeneration to a similar extent. Collectively, our results suggest that the level of Shp2a catalytic activity does not affect the function of Shp2a in the regenerative outgrowth of the zebrafish embryo caudal fin-fold.

179

7

iNtRoduCtioN

Protein tyrosine phosphorylation is at the core of many essential signalling cascades, and the balance between the activity of protein tyrosine kinases and protein tyrosine phosphatases (PTPs) allows for an appropriate cellular response to stimuli (Hunter 2009). In humans, the PTP superfamily consists of 116 cysteine-based enzymes di-

vided into three subgroups based on their structure and function: the classical PTPs, dual-specificity PTPs, and the low molecular weight PTPs (Alonso and Pulido 2016; Tonks 2013). Catalysis by all cysteine-based PTPs is mediated by a conserved cysteine in the PTP domain. The catalytic cysteine residues of PTPs are in the thiolate anion form due to a low pKa, which results from their microenvironment. Reactive oxygen species (ROS), such as H2O2, readily reversibly oxidize cysteine residues that are in the

thiolate anion form, temporarily inactivating the catalytic activity of the PTP (Ostman et al. 2011). Mild oxidation results in conversion of the sulphur atom of the catalytic cysteine from its reduced thiolate anion form (S-_{) to sulphenic acid (SOH), a labile state} that rapidly rearranges to form an intra- or inter-molecular disulphide bond with a

nearby cysteine or a sulphenylamide with an adjacent nitrogen (Salmeen et al. 2003).

In the case of an intramolecular disulphide bond, the nearby cysteine is referred to as

a “back-door cysteine” (Cumming et al. 2004; Sone et al. 1997). Rather unusually, the Src homology 2 (SH2) domain containing phosphatases SHP1 and SHP2 appear to have two back-door cysteines. Under oxidizing conditions, the catalytic cysteine is oxidized

and forms a disulphide bond with one of the two back-door cysteines. However, the

disulphide bond between the catalytic cysteine and one of the back-door cysteines is

rapidly re-reduced, and the two backdoor cysteines form an intramolecular disulphide

bond with each other to stabilize the protein under oxidizing conditions (Chen, Willard, and Rudolph 2009). The disulphide and sulphenylamide bonds are otherwise readily reduced to the catalytically active form of the PTP by endogenous glutathione and thioredoxin systems (Dagnell et al. 2013; Lee et al. 2002; Ostman et al. 2011). Howe-

ver, in extreme oxidizing conditions the PTPs can be hyperoxidized to form irreversible sulphinic acid (SO2H) or sulphonic acid (SO3H) states, permanently inactivating the PTP

(Lo Conte and Carroll 2013a, 2013b).

The classical cytoplasmic PTP SHP2, encoded by the PTPN11 gene, has two SH2

domains, a PTP domain, and a C-terminal domain (Hof et al. 1998), and is readily oxidized by ROS (Karisch et al. 2011; Meng, Fukada, and Tonks 2002). The catalytic cysteine, C459, in the PTP domain is essential for catalytic activity, and mutation to

7

a serine results in an inactive substrate-trapping mutant of SHP2 (Hartman, Schaller, and Agazie 2013; Kolli et al. 2004; Zhang et al. 2004). In addition, the neighbouring arginine, R465, is required for substrate binding and stabilizing the phospho-cysteine intermediate during the catalytic process (Zhang, Wang, and Dixon 1994). The crystal structure of SHP2 shows that SHP2 is in a closed conformation with the N-terminal SH2 domain folding back onto itself and interacting with residues close to the catalytic pocket. In this closed conformation, access of substrates to the catalytic site is blocked, impairing catalytic activity (Hof et al. 1998). Activation of SHP2 is facilitated by dis-

sociation of the SH2 domains from the PTP domain, for instance by binding of the SH2 domains to phospho-tyrosine containing proteins, engendering an open conformation of SHP2, which is able to dephosphorylate its target substrates (Neel, Gu, and Pao 2003). Mutation of some key residues, such as D61 in the N-terminal SH2 domain, also results in an open conformation of SHP2 and increased catalytic activity (Darian et al. 2011). In comparison, mutations of residues close to the catalytic cysteine C459, such as R465 or A461 in the PTP domain, result in reduced catalytic activity (Keilhack et al. 2005; Stewart et al. 2010; Yu et al. 2013).

SHP2 is activated in response to growth factor stimulation, and mediates trans-

duction of mitogen activated kinase (MAPK) and phosphatidylinositide-3-kinase (PI3K) signalling (Wu et al. 2001; Zhang, Liu, and Tu LIU 2002). SHP2 regulates a variety of cellular processes, including adipogenesis (He et al. 2013; Tao et al. 2016), haema-

topoiesis (Chan et al. 2011; Qu et al. 1997), and embryonic development (Bonetti, Rodriguez-Martinez, et al. 2014; Yang et al. 2006), and is mutated in a variety of human diseases (Hendriks et al. 2013). Notably, the activating D61G mutation is commonly found in patients with Noonan Syndrome (NS), whilst the inactivating A461T mutation is commonly found in patients with Noonan Syndrome with Multiple Lentigines (NSML, formerly LEOPARD syndrome). Both mutations result in aberrant MAPK signalling, leading to developmental defects (Araki et al. 2004; Jopling, van Geemen, and den Hertog 2007; Lauriol, Jaffre, and Kontaridis 2015).

ROS are able to coordinate signal transduction in multiple signalling pathways and thereby modulate cellular responses (Finkel 2011; Ray, Huang, and Tsuji 2012; Than-

nickal and Fanburg 2000). Moreover, ROS regulation of the activity of PTPs is evident in a variety of contexts: VE-PTP and PTP-PEST in endothelial cell migration (Frijhoff et al. 2014); PTP1B and TC-PTP in type 2 diabetes mellitus (Gurzov et al. 2014; Meng et al. 2004); and oxidation of various PTPs in cancer (Karisch et al. 2011; Liou and Storz 2010). SHP2 is oxidized following platelet derived growth factor (PDGF) stimulation,

181

7