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Homeostatic scaling of the synapse, such as synaptic down-scaling, has been proposed to offset deleterious effects induced by sustained synaptic strength enhancement. Proper function and subcellular distribution of Src homology 2 domain-containing nonreceptor protein tyrosine phosphatase (SHP2) are required for synaptic plasticity. However, the role of SHP2 in synaptic down-scaling remains largely unknown. Here, using biochemical assays and cell-imaging techniques, we found that synaptic SHP2 levels are temporally regulated during synaptic down-scaling in cultured hippocampal neurons. Furthermore, we observed that a Noonan syndrome-associated mutation of SHP2, resulting in a D61G substitution, prevents synaptic down-scaling. We further show that this effect is due to an inability of the SHP2-D61G variant to properly disassociate from postsynaptic density protein 95, leading to impaired SHP2 dispersion from synaptic sites after synaptic down-scaling. Our findings reveal a molecular mechanism of the Noonan syndrome-associated genetic variant SHP2-D61G that contributes to deficient synaptic down-scaling., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Lu et al.)

Src homology 2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germ line mutations in the PTPN11 gene responsible for catalytic gain or loss of function of SHP2 cause 2 disorders with multiple organ defects: Noonan syndrome (NS) and NS with multiple lentigines (NSML), respectively. Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in 2 mouse models carrying PTPN11 mutations responsible for these 2 syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and αIIbβ3 integrin signaling, platelet secretion, and thromboxane A2 generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice, associated with a significant increase in tail bleeding time. In contrast, NSML mouse platelets exhibited increased platelet activation after GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects, and provides important information for the medical care of patients with NS in situations involving risk of bleeding., (© 2019 by The American Society of Hematology.)

Tyrosyl phosphorylation participates in various pathological and physiological processes, which are regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). The Src homology-2 domain containing phosphatase SHP2 (encoded by PTPN11) is an important phosphatase, which was found to be implicated in the regulation of genetic disease, development, metabolic, neurological, muscle, skeletal disease and cancer. Germline mutations in PTPN11 cause the Noonan Syndrome, LEOPARD syndrome and metachondromatosis. Somatic PTPN11 mutations occur in hematologic malignancies and in solid tumors. SHP2 is also an important component in oncogenic signaling pathways. It may play different roles in different stages and positions of human cancers. Whether SHP2 is an oncogene or cancer suppressor gene remains to be elucidated. Elucidation of the regulatory mechanisms of SHP2 in human disease will provide new insights into disease and new targets for therapy. Here, we summarized the structural basis and recent research progression on SHP2 in various human disease, including genetic and cancer diseases.

Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS.

The RASopathies are a relatively common group of phenotypically similar and genetically related autosomal dominant genetic syndromes caused by missense mutations affecting genes participating in the RAS/mitogen-activated protein kinase (MAPK) pathway that include Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML, formerly LEOPARD syndrome). NS and NSML can be difficult to differentiate during infancy, but the presence of multiple lentigines, café au lait spots, and specific cardiac defects facilitate the diagnosis. Furthermore, individual PTPN11 missense mutations are highly specific to each syndrome and engender opposite biochemical alterations on the function of SHP-2, the protein product of that gene. Here, we report on a 5-year-old male with two de novo PTPN11 mutations in cis, c.1471C>T (p.Pro491Ser), and c.1492C>T (p.Arg498Trp), which are associated with NS and NSML, respectively. This boy's phenotype is intermediate between NS and NSML with facial dysmorphism, short stature, mild global developmental delay, pulmonic stenosis, and deafness but absence of café au lait spots or lentigines. The double-mutant SHP-2 was found to be catalytically impaired. This raises the question of whether clinical differences between NS and NSML can be ascribed solely to the relative SHP-2 catalytic activity., (© 2014 Wiley Periodicals, Inc.)

Germline mutations in PTPN11, encoding Shp2, cause Noonan syndrome (NS) and LEOPARD syndrome (LS), two developmental disorders that are characterized by multiple overlapping symptoms. Interestingly, Shp2 catalytic activity is enhanced by NS mutations and reduced by LS mutations. Defective cardiac development is a prominent symptom of both NS and LS, but how the Shp2 variants affect cardiac development is unclear. Here, we have expressed the most common NS and LS Shp2-variants in zebrafish embryos to investigate their role in cardiac development in vivo. Heart function was impaired in embryos expressing NS and LS variants of Shp2. The cardiac anomalies first occurred during elongation of the heart tube and consisted of reduced cardiomyocyte migration, coupled with impaired leftward heart displacement. Expression of specific laterality markers was randomized in embryos expressing NS and LS variants of Shp2. Ciliogenesis and cilia function in Kupffer's vesicle was impaired, likely accounting for the left/right asymmetry defects. Mitogen-activated protein kinase (MAPK) signaling was activated to a similar extent in embryos expressing NS and LS Shp2 variants. Interestingly, inhibition of MAPK signaling prior to gastrulation rescued cilia length and heart laterality defects. These results suggest that NS and LS Shp2 variant-mediated hyperactivation of MAPK signaling leads to impaired cilia function in Kupffer's vesicle, causing left-right asymmetry defects and defective early cardiac development.

Here, we describe neurobehavioral features in patients with RASopathies (i.e., Noonan syndrome, LEOPARD syndrome, Costello syndrome, and cardiofaciocutaneous syndrome), developmental disorders caused by mutations in genes coding transducers participating in the RAS-MAPK signaling cascade. Parents of 70 individuals with a RASopathy were asked to fill out the following questionnaires: Child Behavior Checklist (CBCL), Social Communication Questionnaire version lifetime (SCQ-L), and Modified Checklist for Autism in toddlers (M-CHAT). Data analysis indicated high rates of internalizing (37%) and externalizing problems (31%) on CBCL. Scores over the cut-off were documented in 64% of patients with cardiofaciocutaneous syndrome, 44% with Costello syndrome, and 12% with Noonan syndrome on SCQ-L/M-CHAT. Our findings indicate that mutations promoting dysregulation of the RAS-MAPK cascade mark an increased psychopathological risk and highlight that autistic-like behavior could be underdiagnosed in patients with RASopathies., (© 2014 Wiley Periodicals, Inc.)

Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the human PTPN11 gene, is a ubiquitously expressed protein tyrosine phosphatase (PTP) that consists of two tandem Src homology (SH2) domains (N-SH2 and C-SH2), a PTP catalytic domain, and a C-terminal tail with tyrosyl phosphorylation sites. It plays critical roles in numerous cellular processes through the regulation of various signaling pathways in PTP catalytic activity-dependent and -independent manners. Dysfunction of SHP2 resulting from pathogenic mutations and aberrant expression leads to the dysregulation of multiple signaling pathways, thus contributing to different human disorders. Germline and somatic mutations in PTPN11 are involved in Noonan syndrome (NS), LEOPARD syndrome (LS), and hematological malignancies, as well as several solid tumors. In this report, we provide an overview of the current knowledge of the structure and function of SHP2, and further discuss the molecular and pathogenic mechanism of SHP2 in human diseases, with a special focus on tumorigenesis. Furthermore, we summarize that SHP2 might itself represent a potential drug target for cancer prevention and treatment. Ongoing research and development of SHP2-specific inhibitors would enhance this potential.

Background: Noonan syndrome is an autosomal, dominantly inherited disease; it is physically characterized by short stature, short neck, webbed neck, abnormal auricles, high arched palate, and cardiovascular malformation. Its pathological condition is thought to be due to a gain-of-function mutation in the Ras-mitogen-activated protein kinase (MAPK) signal transduction pathway. Eyelid abnormalities such as ocular hypertelorism and blepharoptosis are the most commonly observed eye complications., Case Presentation: We report a case of Noonan syndrome associated with mature cataract that required operation. A 42-year-old man was diagnosed with Noonan syndrome at the age of 1 year. He underwent an eye examination after complaining of decreased visual acuity in the right eye and was diagnosed with mature cataract, which was treated by cataract surgery. There were no intraoperative complications, and the postoperative course was uneventful. Protein analysis of lens capsule and epithelium at capsulorhexis showed MAPK cascade proteins such as ERK and p38MAPK were upregulated. An abnormality in the PTPN11 gene was also observed; a potential mechanism of cataract onset may be that opacity of the lens rapidly progressed due to abnormal activation of the Ras-MAPK signal transduction pathway., Conclusion: This case highlights the possible association of cataract formation with MAPK cascade protein upregulation in Noonan syndrome.

SHP2/PTPN11 is a key regulator of cytokine, growth factor and integrin signaling. SHP2 influences cell survival, proliferation and differentiation by regulating major signaling pathways. Mutations in PTPN11 cause severe diseases like Noonan, LEOPARD syndrome or leukemia. Whereas several of these mutations result in altered enzymatic activity due to impaired auto-inhibition, not all disease patterns can be explained by this mechanism. In this study we analyzed altered binding properties of disease-related SHP2-mutants bearing point mutations within the SH2-domain (T42A, E139D, and R138Q). Mutants were chosen according to SPR assays, which revealed different binding properties of mutated SH2 towards phosphorylated receptor peptides. To analyze global changes in mutant binding properties we applied quantitative mass spectrometry (SILAC). Using an in vitro approach we identified overall more than 1000 protein candidates, which specifically bind to the SH2-domain of SHP2. We discovered that mutations in the SH2-domain selectively affected protein enrichment by altering the binding capacity of the SH2-domain. Mutation-dependent, enhanced or reduced exposure of SHP2 to its binding partners could have an impact on the dynamics of signaling networks. Thus, disease-associated mutants of SHP2 should not only be discussed in the context of deregulated auto-inhibition but also with respect to deregulated protein targeting of the SHP2 mutants., Biological Significance: Using quantitative mass spectrometry based proteomics we provided evidence that disease related mutations in SHP2 domains of SHP2 are able to influence SHP2 recruitment to its targets in mutation dependent manner. We discovered that mutations in the SH2-domain selectively affected protein enrichment ratios suggesting altered binding properties of the SH2-domain. We demonstrated that mutations within SHP2, which had been attributed to affect the enzymatic activity (i.e. affect the open/close status of SHP2), also differ in respect to binding properties. Our study indicates that SHP2 mutations need to be discussed not only in terms of deregulated auto-inhibition but also with respect to deregulated protein targeting properties of the SHP2 mutants. Discovery of the new binding partners for disease-related SHP2 mutants might provide a fruitful foundation for developing strategies targeting Noonan-associated leukemia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.

Noonan syndrome (NS), a genetic disease caused in half of cases by activating mutations of the tyrosine phosphatase SHP2 (PTPN11), is characterized by congenital cardiopathies, facial dysmorphic features, and short stature. How mutated SHP2 induces growth retardation remains poorly understood. We report here that early postnatal growth delay is associated with low levels of insulin-like growth factor 1 (IGF-1) in a mouse model of NS expressing the D61G mutant of SHP2. Conversely, inhibition of SHP2 expression in growth hormone (GH)-responsive cell lines results in increased IGF-1 release upon GH stimulation. SHP2-deficient cells display decreased ERK1/2 phosphorylation and rat sarcoma (RAS) activation in response to GH, whereas expression of NS-associated SHP2 mutants results in ERK1/2 hyperactivation in vitro and in vivo. RAS/ERK1/2 inhibition in SHP2-deficient cells correlates with impaired dephosphorylation of the adaptor Grb2-associated binder-1 (GAB1) on its RAS GTPase-activating protein (RASGAP) binding sites and is rescued by interfering with RASGAP recruitment or function. We demonstrate that inhibition of ERK1/2 activation results in an increase of IGF-1 levels in vitro and in vivo, which is associated with significant growth improvement in NS mice. In conclusion, NS-causing SHP2 mutants inhibit GH-induced IGF-1 release through RAS/ERK1/2 hyperactivation, a mechanism that could contribute to growth retardation. This finding suggests that, in addition to its previously shown beneficial effect on NS-linked cardiac and craniofacial defects, RAS/ERK1/2 modulation could also alleviate the short stature phenotype in NS caused by PTPN11 mutations.

Noonan syndrome is one of the most common causes of human congenital heart disease and is frequently associated with missense mutations in the protein phosphatase SHP-2. Interestingly, patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), juvenile myelomonocytic leukemia (JMML) and LEOPARD syndrome frequently carry a second, somatically introduced subset of missense mutations in SHP-2. To determine the cellular and molecular mechanisms by which SHP-2 regulates heart development and, thus, understand how Noonan-associated mutations affect cardiogenesis, we introduced SHP-2 encoding the most prevalent Noonan syndrome and JMML mutations into Xenopus embryos. Resulting embryos show a direct relationship between a Noonan SHP-2 mutation and its ability to cause cardiac defects in Xenopus; embryos expressing Noonan SHP-2 mutations exhibit morphologically abnormal hearts, whereas those expressing an SHP-2 JMML-associated mutation do not. Our studies indicate that the cardiac defects associated with the introduction of the Noonan-associated SHP-2 mutations are coupled with a delay or arrest of the cardiac cell cycle in M-phase and a failure of cardiomyocyte progenitors to incorporate into the developing heart. We show that these defects are a result of an underlying malformation in the formation and polarity of cardiac actin fibers and F-actin deposition. We show that these defects can be rescued in culture and in embryos through the inhibition of the Rho-associated, coiled-coil-containing protein kinase 1 (ROCK), thus demonstrating a direct relationship between SHP-2(N308D) and ROCK activation in the developing heart.

Reports on Noonan syndrome (NS) have documented multiple types of coagulation defects and bleeding diathesis, and a wide range of clinical presentations. Early studies suggested that a large proportion of NS patients have coagulation defects, whereas more recent reports indicate low rates of coagulopathy. The aim of this study was to evaluate phenotypic characteristics, PTPN11 gene mutations, and hematological and coagulation parameters in 30 clinically diagnosed cases of NS. One of the NS patients had a history of easy bruising; however, his hematological and coagulation tests were normal. None of the other patients had clinical coagulation problems. In the NS group, values for platelet count, activity of factors XI, XII, and protein C were significantly lower than the corresponding means for the control group. However, the results of coagulation tests in the NS group were diagnostically inconclusive and only one patient had clinical signs of coagulopathy. Interestingly, two NS patients had low protein C activity. One of these children had an A1517C mutation and transient myelodysplasia. The other patient had a C1528G mutation in exon 13 that has not been reported previously. Neither of these individuals experienced a thrombotic event or any complication during approximately 3 years of follow-up. For all patients clinically diagnosed with NS, a thorough history of coagulation issues should be taken and first-line coagulation testing should be done to evaluate for bleeding diathesis. However, if these assessments reveal nothing abnormal, complications related to coagulation are unlikely and extensive testing is unnecessary., (© 2010 Wiley-Liss, Inc.)

Noonan syndrome (NS; OMIM 163950) is an autosomal dominant disorder with variable clinical expression and genetic heterogeneity. Clinical manifestations include characteristic facial features, short stature, and cardiac anomalies. Mutations in protein-tyrosine phosphatase, non-receptor-type 11 (PTPN11), encoding SHP-2, account for about half of NS patients. We report on a Moroccan family with two children with NS and apparently unaffected parents. The molecular studies showed the heterozygous mutation c.922A>G of PTPN11 gene in the two affected sibs. Neither the parents, nor the oldest brother carries this mutation in hematologic cells. The mutation was also absent in buccal epithelial cells and fingernails of both parents. We believe this is the first report of germ cell mosaicism in NS and suggest an empirical risk for recurrence of that is less than 1%., (© 2010 Wiley-Liss, Inc.)

Objective: To investigate the mutations in protein tyrosine phosphatase, nonreceptor-type 11 (PTPN11) gene in patients with Noonan syndrome (NS)., Methods: Three sporadic patients with NS were studied. Genomic DNAs were extracted from peripheral blood leukocytes. All 15 coding exons and their flanking intronic boundaries of the PTPN11 gene were amplified by polymerase chain reaction and followed by direct sequencing. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their affected child. The identified mutation was screened in 100 healthy individuals for exclusion of polymorphism by restriction endonuclease digestion of the PCR products. Protein conservation analysis was performed among 10 species using an online ClustalW tool., Results: Direct DNA sequence analysis identified a heterozygous 181G to A change in exon 3 of the PTPN11 gene in one patient, which resulted in the substitution of an aspartic acid residue by an asparagine at codon 61. The mutation was absent in his parents and 100 controls, and is located in a highly conserved amino acid site. No mutation in the coding region of PTPN11 gene was observed in the other two patients., Conclusion: The p.D61N mutation was reported previously in Caucasians and is a de-novo mutation in this patient. Our study further confirmed that the p.D61N is a pathogenic mutation for NS and consistent with the clinical diagnosis. Additional genes may be involved in the other two patients with NS, indicating high genetic heterogeneity of this disease.

Noonan syndrome (NS) is an autosomal dominant disorder, and a main feature is congenital heart malformation. About 50% of cases are caused by gain-of-function mutations in the tyrosine phosphatase SHP2/PTPN11, a downstream regulator of ERK/MAPK. Recently it was reported that SHP2 also localizes to the mitochondrial intercristae/intermembrane space (IMS), but the role of SHP2 in mitochondria is unclear. The mitochondrial oxidative phosphorylation (OxPhos) system provides the vast majority of cellular energy and produces reactive oxygen species (ROS). Changes in ROS may interfere with organ development such as that observed in NS patients. Several phosphorylation sites have been found in OxPhos components including cytochrome c oxidase (CcO) and cytochrome c (Cytc), and we hypothesized that OxPhos complexes may be direct or indirect targets of SHP2. We analyzed mitochondrial function using mouse fibroblasts from wild-types, SHP2 knockdowns, and D61G SHP2 mutants leading to constitutively active SHP2, as found in NS patients. Levels of OxPhos complexes were similar except for CcO and Cytc, which were 37% and 28% reduced in the D61G cells. However, CcO activity was significantly increased, as we also found for two lymphoblast cell lines from NS patients with two independent mutations in PTPN11. D61G cells showed lower mitochondrial membrane potential and 30% lower ATP content compared to controls. ROS were significantly increased; aconitase activity, a marker for ROS-induced damage, was decreased; and catalase activity was increased in D61G cells. We propose that decreased energy levels and/or increased ROS may explain, at least in part, some of the clinical features in NS that overlap with children with mitochondrial disorders., (Copyright 2009 Elsevier B.V. All rights reserved.)

Short stature, with a mean final height almost two standard deviations below the normal mean, is a major feature of Noonan syndrome. The biological basis of the growth failure is not yet clear. The recent detection of mutations in the protein tyrosine phosphatase, non-receptor type 11 gene (PTPN11) in half of all individuals with Noonan syndrome has opened up a new perspective from the endocrine point of view, since the tyrosine phosphatase SHP2 encoded by PTPN11 is implicated in the downregulation of growth hormone (GH) receptor signalling. Current data show decreased insulin-like growth factor (IGF)-I and IGF-binding protein 3 (IGFBP-3) levels in those children with Noonan syndrome who carry PTPN11 mutations. GH responsiveness seems to be reduced in the presence of PTPN11 mutations, but, so far, data are too scarce to draw any final conclusions. Children with Noonan or Noonan-related syndromes carrying mutations in components of the Ras-mitogen-activated protein kinase (MAPK) signalling pathway downstream from SHP2 also have short stature, though less frequently in the case of SOS1 mutations. Therefore, apart from the disturbance of GH signalling, there must be other relevant mechanisms that influence longitudinal growth in Noonan syndrome. In a small subgroup of patients with Noonan syndrome and Noonan-related syndromes, tumour risk is increased. This susceptibility is relevant when GH therapy is considered. Progress in the understanding of cell regulation by Ras-MAPK signalling and its interconnection with other pathways will hopefully provide evidence on which therapy might be helpful and which might be nocuous in the care of children with Noonan syndrome., (Copyright 2009 S. Karger AG, Basel.)

Noonan syndrome (NS), the most common single-gene cause of congenital heart disease, is an autosomal dominant disorder that also features proportionate short stature, facial abnormalities, and an increased risk of myeloproliferative disease. Germline-activating mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, cause about half of NS cases; other causative alleles include KRAS, SOS1, and RAF1 mutants. We showed previously that knock-in mice bearing the NS mutant Ptpn11(D61G) on a mixed 129S4/SvJae X C57BL6/J background exhibit all major NS features, including a variety of cardiac defects, with variable penetrance. However, the cellular and molecular mechanisms underlying NS cardiac defects and whether genetic background and/or the specific NS mutation contribute to the NS phenotype remained unclear. Here, using an inducible knock-in approach, we show that all cardiac defects in NS result from mutant Shp2 expression in the endocardium, not in the myocardium or neural crest. Furthermore, the penetrance of NS defects is affected by genetic background and the specific Ptpn11 allele. Finally, ex vivo assays and pharmacological approaches show that NS mutants cause cardiac valve defects by increasing Erk MAPK activation, probably downstream of ErbB family receptor tyrosine kinases, extending the interval during which cardiac endocardial cells undergo endocardial-mesenchymal transformation. Our data provide a mechanistic underpinning for the cardiac defects in this disorder.

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced beta1 integrin outside-in signaling. In response to beta1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.

Missense mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, cause clinically similar but distinctive disorders, LEOPARD (LS) and Noonan (NS) syndromes. The LS is an autosomal dominant disorder with pleomorphic developmental abnormalities including lentigines, cardiac defects, short stature and deafness. Biochemical analyses indicated that LS alleles engender loss-of-function (LOF) effects, while NS mutations result in gain-of-function (GOF). These biochemical findings lead to an enigma that how PTPN11 mutations with opposite effects on function result in disorders that are so similar. To study the developmental effects of the commonest LS PTPN11 alleles (Y279C and T468M), we generated LS transgenic fruitflies using corkscrew (csw), the Drosophila orthologue of PTPN11. Ubiquitous expression of the LS csw mutant alleles resulted in ectopic wing veins and, for the Y279C allele, rough eyes with increased R7 photoreceptor numbers. These were GOF phenotypes mediated by increased RAS/MAPK signaling and requiring the LS mutant's residual phosphatase activity. Our findings provide the first evidence that LS mutant alleles have GOF developmental effects despite reduced phosphatase activity, providing a rationale for how PTPN11 mutations with GOF and LOF produce similar but distinctive syndromes.

We describe the previously unreported condition of Hodgkin's lymphoma in a patient with Noonan syndrome caused by germ-line mutations (1507G > C, Gly503Arg) in exon 13 of the PTPN11 gene. PTPN11, encoding SHP-2, is the first identified gene for Noonan syndrome and also the first identified proto-oncogene that encodes a tyrosine phosphatase. This somatic mutation has ever been reported in juvenile myelomonocytic leukemia (JMML). Furthermore, the functional analysis of this mutant SHP-2 has shown it to have enhanced phosphatase activity. Mutational analysis of PTPN11 gene in cancer cells and understanding how SHP-2 contributes to oncogenesis will provide new insight into the pathogenesis of Hodgkin's lymphoma.

Noonan syndrome (NS) is an autosomal dominant disorder that is associated with multiple developmental abnormalities. Activated mutations of the protein-tyrosine phosphatase, SHP-2/PTPN11, have been reported in approximately 50% of NS cases. Despite being activated, NS-associated SHP-2 mutants require plasma membrane proximity to evoke disease-associated signaling. Here we show that NS-associated SHP-2 mutants induce hypertyrosyl phosphorylation of the transmembrane glycoproteins, SIRPalpha (signal-regulatory protein alpha) and PZR (protein zero-related), resulting in their increased association with NS-associated SHP-2 mutants. NS-associated SHP-2 mutants enhanced SIRPalpha and PZR tyrosyl phosphorylation either by impairing SIRPalpha dephosphorylation or by promoting PZR tyrosyl phosphorylation. Importantly, during embryogenesis in a mouse model of NS, SIRPalpha and PZR were hypertyrosyl-phosphorylated and bound increased levels of the NS-associated SHP-2 mutant. SIRPalpha and PZR have been implicated in extracellular matrix-dependent signaling. Mouse embryonic fibroblasts derived from a mouse model of NS displayed enhanced ERK activation in response to fibronectin plating. Knockdown of SIRPalpha and PZR in these cells attenuated the enhanced activation of ERK following fibronectin plating. Thus, SIRPalpha and PZR serve as scaffolds that facilitate plasma membrane recruitment and signaling of NS-associated SHP-2 mutants.

Background: Heart failure is the leading cause of death in the United States. By delineating the pathways that regulate cardiomyocyte function, we can better understand the pathogenesis of cardiac disease. Many cardiomyocyte signaling pathways activate protein tyrosine kinases. However, the role of specific protein tyrosine phosphatases (PTPs) in these pathways is unknown., Methods and Results: Here, we show that mice with muscle-specific deletion of Ptpn11, the gene encoding the SH2 domain-containing PTP Shp2, rapidly develop a compensated dilated cardiomyopathy without an intervening hypertrophic phase, with signs of cardiac dysfunction appearing by the second postnatal month. Shp2-deficient primary cardiomyocytes are defective in extracellular signal-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) activation in response to a variety of soluble agonists and pressure overload but show hyperactivation of the RhoA signaling pathway. Treatment of primary cardiomyocytes with Erk1/2- and RhoA pathway-specific inhibitors suggests that both abnormal Erk/MAPK and RhoA activities contribute to the dilated phenotype of Shp2-deficient hearts., Conclusions: Our results identify Shp2 as the first PTP with a critical role in adult cardiac function, indicate that in the absence of Shp2 cardiac hypertrophy does not occur in response to pressure overload, and demonstrate that the cardioprotective role of Shp2 is mediated via control of both the Erk/MAPK and RhoA signaling pathways.

Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Normal development of the nervous system relies on the spatially and temporally well-controlled differentiation of neurons and glia. Here, we discuss the intra- and extracellular molecular mechanisms that underlie the sequential genesis of neurons and glia, emphasizing recent studies describing the role of a signaling molecule, the tyrosine phosphatase SHP2, in normal brain development. Activation of SHP2 simultaneously enhances downstream activation of the MEK-ERK pathway, which subsequently promotes neurogenesis, while inhibiting the JAK-STAT pathway, which is critical for astroglial differentiation. Mutations in SHP2 that increase its tyrosine phosphatase activity cause a mental retardation-related disorder, Noonan syndrome. An imbalance in neurogenesis versus gliogenesis due to SHP2 mutations may contribute to Noonan syndrome.

Noonan syndrome (NS) is an autosomal dominant disorder characterized by a wide spectrum of defects, which most frequently include proportionate short stature, craniofacial anomalies, and congenital heart disease (CHD). NS is the most common nonchromosomal cause of CHD, and 80%-90% of NS patients have cardiac involvement. Mutations within the protein tyrosine phosphatase Src homology region 2, phosphatase 2 (SHP2) are responsible for approximately 50% of the cases of NS with cardiac involvement. To understand the developmental stage- and cell type-specific consequences of the NS SHP2 gain-of-function mutation, Q79R, we generated transgenic mice in which the mutated protein was expressed during gestation or following birth in cardiomyocytes. Q79R SHP2 embryonic hearts showed altered cardiomyocyte cell cycling, ventricular noncompaction, and ventricular septal defects, while, in the postnatal cardiomyocyte, Q79R SHP2 expression was completely benign. Fetal expression of Q79R led to the specific activation of the ERK1/2 pathway, and breeding of the Q79R transgenics into ERK1/2-null backgrounds confirmed the pathway's necessity and sufficiency in mediating mutant SHP2's effects. Our data establish the developmental stage-specific effects of Q79R cardiac expression in NS; show that ablation of subsequent ERK1/2 activation prevents the development of cardiac abnormalities; and suggest that ERK1/2 modulation could have important implications for developing therapeutic strategies in CHD.

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.

Human growth is a highly complicated process, but it is obviously influenced by a genetic factor. Recent genome-wide linkage analyses suggested some genetic regions underlying stature variations. However, any specific genes underlying stature variations have not been identified. Noonan syndrome (NS) is an autosomal dominant disorder clinically characterized by short stature, minor facial anomalies, and congenital heart defects. Recently, PTPN11 (protein-tyrosine phosphatase, nonreceptor-type 11) has been identified as a major responsible gene for NS, causing about half of the affected individuals. We herein report a large family demonstrating NS caused by one of the common PTPN11 mutations, c.188 A > G (Y63C). In this family, the patients were apparently healthy, but heterozygosity of the c.188 A > G (Y63C) mutation was related to growth impairment. This finding suggested that PTPN11 genetic variants contribute to adult height in the general population. However, c.188 A > G (Y63C) was not identified in 96 short individuals from the general population of 2,281 healthy adults. Thus, it is unlikely that PTPN11 is one of the genes underlying stature variations in the general population.

Reversible protein tyrosyl phosphorylation of cell surface receptors and downstream intracellular transducers is a major regulatory mechanism used to modulate cellular responses to extracellular stimuli, and its deregulation frequently drives aberrant cell proliferation, survival and/or differentiation. SHP-2 is a cytoplasmic Src-homology 2 domain-containing protein tyrosine phosphatase that plays an important role in intracellular signaling and is required during development and hematopoiesis. Germ-line missense mutations in PTPN11, the gene coding SHP-2, have been discovered as a major molecular event underlying Noonan syndrome, an autosomal dominant trait characterized by short stature, dysmorphic facies, and congenital heart defects, as well as in other closely related developmental disorders. More recently, a distinct class of missense mutations in the same gene has been identified to occur as a somatic event contributing to myeloid and lymphoid malignancies. This review focuses on the role of SHP-2 in signal transduction, development and hematopoiesis, as well as on the consequences of SHP-2 gain-of-function.

LEOPARD syndrome (lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) is an autosomal dominant condition. The main clinical features include multiple lentigines, cardiovascular defects, and facial anomalies, some of which are shared with Noonan syndrome (NS). Recent reports have shown that LEOPARD syndrome can be caused by mutations in PTPN11, the gene in which mutations can produce NS. Here we report the findings of mutation screening and linkage analysis of PTPN11 in three families with LEOPARD syndrome. We identified a novel mutation in one family. The mutation (1529A>C) substitutes proline for glutamine at amino acid 510 (Gln510Pro). No variations in sequence were observed in the other two families, and negative LOD scores excluded linkage to the PTPN11 locus, showing that LEOPARD syndrome is genetically heterogeneous.

Noonan syndrome was recently reported to be caused by mutations in the PTPN11 gene in 40% of the cases. This gene encodes the nonreceptor-type protein tyrosine phosphatase SHP-2 and has been shown to be self down-regulated with the concurrency of two SH2 domains. Insertion of a specific loop (D'EF) from N-terminal SH2 domain into the SHP-2 active-site is responsible for the reversible inhibition of the phosphatase activity. Here we report the first in frame trinucleotide deletion resulting in the removal of Aspartate 61 (D61del), a key residue of the N-terminal SH2 D'EF loop. Energetic-based structural analysis and electrostatic calculations carried out on the wild-type and mutant proteins predict lower stability of the D'EF loop for the D61del variant as compared to the wild type indicating better access to the active site and most likely an enzyme activated for longer extent. Similar computations were performed on the previously functionally characterized gain-of-function D61Y mutant and similar behaviors were observed. The simulation data for the D61del and D61Y mutants suggest that both variants could yield more catalytic cycles than the wild-type molecule in the same timespan because of the opening of the active site. It also supports the notion that D61 plays a major role for proper down-regulation of the protein tyrosine phosphatase activity of SHP-2., ((c) 2004 Wiley-Liss, Inc.)

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

Noonan syndrome is a developmental disorder with dysmorphic facies, short stature, cardiac defects, and skeletal anomalies, which can be caused by missense PTPN11 mutations. PTPN11 encodes Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2 or SHP-2), a protein tyrosine phosphatase that acts in signal transduction downstream to growth factor, hormone, and cytokine receptors. We compared the functional effects of three Noonan syndrome-causative PTPN11 mutations on SHP2's phosphatase activity, interaction with a binding partner, and signal transduction. All SHP2 mutants had significantly increased basal phosphatase activity compared to wild type, but that activity varied significantly between mutants and was further increased after epidermal growth factor stimulation. Cells expressing SHP2 mutants had prolonged extracellular signal-regulated kinase 2 activation, which was ligand-dependent. Binding of SHP2 mutants to Grb2-associated binder-1 was increased and sustained, and tyrosine phosphorylation of both proteins was prolonged. Coexpression of Grb2-associated binder-1-FF, which lacks SHP2 binding motifs, blocked the epidermal growth factor-mediated increase in SHP2's phosphatase activity and resulted in a dramatic reduction of extracellular signal-regulated kinase 2 activation. Taken together, these results document that Noonan syndrome-associated PTPN11 mutations increase SHP2's basal phosphatase activity, with greater activation when residues directly involved in binding at the interface between the N-terminal Src homology 2 and protein tyrosine phosphatase domains are altered. The SHP2 mutants prolonged signal flux through the RAS/mitogen-activated protein kinase (ERK2/MAPK1) pathway in a ligand-dependent manner that required docking through Grb2-associated binder-1 (GAB1), leading to increased cell proliferation., (Copyright 2004 Wiley-Liss, Inc.)

We report here that individuals with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) have germline mutations in PTPN11 and that somatic mutations in PTPN11 account for 34% of non-syndromic JMML. Furthermore, we found mutations in PTPN11 in a small percentage of individuals with myelodysplastic syndrome (MDS) and de novo acute myeloid leukemia (AML). Functional analyses documented that the two most common mutations in PTPN11 associated with JMML caused a gain of function.

We surveyed 16 subjects with the clinical diagnosis of Noonan Syndrome (NS1) from 12 families and their relevant family members for mutations in PTPN11/SHP2 using direct DNA sequencing. We found three different mutations among five families. Two unrelated subjects shared the same de novo missense substitution in exon 13 (S502T); an additional two unrelated families had a mutation in exon 3 (Y63C); and one subject had the amino acid substitution Y62D, also in exon 3. None of the three mutations were present in ethnically matched controls. In the mature protein model, the exon 3 mutants and the exon 13 mutant amino acids cluster at the interface between the N' SH2 domain and the phosphatase catalytic domain. Six of eight subjects with PTPN11/SHP2 mutations had pulmonary valve stenosis while no mutations were identified in those subjects (N = 4) with hypertrophic cardiomyopathy. An additional four subjects with possible Noonan syndrome were evaluated, but no mutations in PTPN11/SHP2 were identified. These results confirm that mutations in PTPN11/SHP2 underlie a common form of Noonan syndrome, and that the disease exhibits both allelic and locus heterogeneity. The observation of recurrent mutations supports the hypothesis that a special class of gain-of-function mutations in SHP2 give rise to Noonan syndrome., (Copyright 2002 Wiley-Liss, Inc.)

Noonan syndrome (NS) is a developmental disorder characterized by facial dysmorphia, short stature, cardiac defects, and skeletal malformations. We recently demonstrated that mutations in PTPN11, the gene encoding the non-receptor-type protein tyrosine phosphatase SHP-2 (src homology region 2-domain phosphatase-2), cause NS, accounting for approximately 50% of cases of this genetically heterogeneous disorder in a small cohort. All mutations were missense changes and clustered at the interacting portions of the amino-terminal src-homology 2 (N-SH2) and protein tyrosine phosphatase (PTP) domains. A gain of function was postulated as a mechanism for the disease. Here, we report the spectrum and distribution of PTPN11 mutations in a large, well-characterized cohort with NS. Mutations were found in 54 of 119 (45%) unrelated individuals with sporadic or familial NS. There was a significantly higher prevalence of mutations among familial cases than among sporadic ones. All defects were missense, and several were recurrent. The vast majority of mutations altered amino acid residues located in or around the interacting surfaces of the N-SH2 and PTP domains, but defects also affected residues in the C-SH2 domain, as well as in the peptide linking the N-SH2 and C-SH2 domains. Genotype-phenotype analysis revealed that pulmonic stenosis was more prevalent among the group of subjects with NS who had PTPN11 mutations than it was in the group without them (70.6% vs. 46.2%; P<.01), whereas hypertrophic cardiomyopathy was less prevalent among those with PTPN11 mutations (5.9% vs. 26.2%; P<.005). The prevalence of other congenital heart malformations, short stature, pectus deformity, cryptorchidism, and developmental delay did not differ between the two groups. A PTPN11 mutation was identified in a family inheriting Noonan-like/multiple giant-cell lesion syndrome, extending the phenotypic range of disease associated with this gene.

Noonan syndrome (MIM 163950) is an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature and heart disease (most commonly pulmonic stenosis and hypertrophic cardiomyopathy). Webbed neck, chest deformity, cryptorchidism, mental retardation and bleeding diatheses also are frequently associated with this disease. This syndrome is relatively common, with an estimated incidence of 1 in 1,000-2,500 live births. It has been mapped to a 5-cM region (NS1) [corrected] on chromosome 12q24.1, and genetic heterogeneity has also been documented. Here we show that missense mutations in PTPN11 (MIM 176876)-a gene encoding the nonreceptor protein tyrosine phosphatase SHP-2, which contains two Src homology 2 (SH2) domains-cause Noonan syndrome and account for more than 50% of the cases that we examined. All PTPN11 missense mutations cluster in interacting portions of the amino N-SH2 domain and the phosphotyrosine phosphatase domains, which are involved in switching the protein between its inactive and active conformations. An energetics-based structural analysis of two N-SH2 mutants indicates that in these mutants there may be a significant shift of the equilibrium favoring the active conformation. This implies that they are gain-of-function changes and that the pathogenesis of Noonan syndrome arises from excessive SHP-2 activity.