Diverse Biochemical Properties of Shp2 Mutants

Abstract

Mutations in the Src homology 2 (SH2)-containing protein-tyrosine phosphatase Shp2 (PTPN11) underlie half of the cases of the autosomal dominant genetic disorder Noonan syndrome, and somatic Shp2 mutations are found in several hematologic and solid malignancies. Earlier studies of small numbers of mutants suggested that disease-associated mutations cause constitutive (SH2 binding-independent) activation and that cancer-associated mutants are more active than those associated with Noonan syndrome. We have characterized a larger panel of Shp2 mutants and find that this "activity-centric" model cannot explain the behaviors of all pathogenic Shp2 mutations. Instead, enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees. Furthermore, there is no absolute correlation between the mutants' extents of basal activation and the diseases they induce. We propose that activated mutants of Shp2 modulate signaling from specific stimuli to a subset of effectors and provide a theoretical framework for understanding the complex relationship between Shp2 activation, intracellular signaling, and pathology.

Reduced phosphatase activity of SHP‐2 in LEOPARD syndrome: Consequences for PI3K binding on Gab1

“…As additional controls, we verified that the well-characterized catalytically dead C459G construct displayed no PTP activity, and that the leukemia-associated D61Y mutant and of the NS-associated D61del mutant have an increased PTP activity compared to WT-SHP-2. These two mutants are not significantly overstimulated by the IRS1-pY1172 peptide in these conditions, but this is in agreement with a previous report [8]. In addition, when the immunocomplexes were incubated with the activating peptide alone, the release of phosphate was undetectable for any of SHP-2 construct (data not shown), indicating that this phosphopeptide was not significantly dephosphorylated by SHP-2 when used in these conditions appropriate to observe its activating effect.…”

“…Following immunoprecipitation, the PTP activity of the different constructs was assayed using as substrate a synthetic phosphopeptide encompassing Y529 of Src, a standard in vitro substrate of PTP [3,9]. To test these mutants under stimulation by an upstream SHP-2 activator, the immunocomplexes were also incubated with 10 lM of a phosphopeptide containing the SHP-2-binding site of IRS-1 (IRS1-pY1172) [8]. As shown in Fig.…”

Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors

“…The results of the substrate-trapping experiment revealed that DM/T507K SHP-2-Myc specifically coprecipitated several tyrosinephosphorylated proteins with molecular masses of approximately 65 000B70 000 Da, which were not coprecipitated with DM SHP-2-Myc or DM/E76K SHP-2-Myc ( Figure 3b, lanes 5-7). We also note that the substrate specificity of DM/E76K SHP-2-Myc did not differ from that of DM SHP-2-Myc, indicating that E76K is a hypermorphic but not neomorphic SHP-2 mutation as has been suggested (Keilhack et al, 2005) ( Figure 3b). From these results, we concluded that T507K SHP-2 exhibits altered substrate specificity either through acquisition of new substrates or significant alterations in its interaction with particular substrates.…”

“…In this phosphatase assay, the E76K mutant of SHP-2, the most common leukemia-specific mutant that causes complete relief of autoinhibition and full activation (Neel et al, 2003;Keilhack et al, 2005;Tartaglia et al, 2006), exhibited more than 100-fold activity of wild-type SHP-2. In contrast, the T507K mutant showed only twofold increase in phosphatase activity compared with that of wild-type SHP-2 ( Figure 2b).…”

“…Furthermore, the observation that approximately 25% of JMML cases possess oncogenic RAS mutation indicates that deregulation of the RAS-effector pathways through gain-of-function mutation of RAS or SHP-2 plays a causative role in the development of JMML (Lauchle et al, 2006). Intriguingly, the individual amino-acid substitutions of SHP-2 identified in Noonan syndrome and sporadic JMML hardly overlap each other , and leukemia (JMML)-specific SHP-2 mutants generally exhibit highly elevated basal phosphatase activity compared with the basal phosphatase level of wild type or Noonan-specific SHP-2 (Tartaglia et al, 2003Keilhack et al, 2005).…”