Shp2 Knockdown and Noonan/LEOPARD Mutant Shp2–Induced Gastrulation Defects

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.     

Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects

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.     

The 'Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling

Src homology-2 (SH2) domain-containing phosphatases (Shps) are a small, highly conserved subfamily of protein-tyrosine phosphatases, members of which are present in both vertebrates and invertebrates. The mechanism of regulation of Shps by ligand binding is now well understood. Much is also known about the normal signaling pathways regulated by each Shp and the consequences of Shp deficiency. Recent studies have identified mutations in human Shp2 as the cause of the inherited disorder Noonan syndrome. Shp2 mutations might also contribute to the pathogenesis of some leukemias. In addition, Shp2 might be a key virulence determinant for the important human pathogen Helicobacter pylori. Despite these efforts, however, the key targets of each Shp have remained elusive. Identifying these substrates remains a major challenge for future research.     

PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects

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.     

New Approaches to Prevent LEOPARD Syndrome-associated Cardiac Hypertrophy by Specifically Targeting Shp2-dependent Signaling

In LEOPARD syndrome (LS) patients, mutations in the protein tyrosine phosphatase Shp2 cause hypertrophic cardiomyopathy. The prohypertrophic effects of mutant Shp2 are mediated downstream by hyperactivation of mammalian target of rapamycin. Our goal was to further define the signaling cascade that is essential for the underlying pathomechanism, thus expanding the list of potential future therapeutic targets.Using cultured neonatal rat cardiomyocytes with adenoviral gene delivery and pharmacological inhibitors, we found that hypertrophy induced by a particularly aggressive LS mutation in Shp2 depends on hyperactivation of Akt and focal adhesion kinase as well as mammalian target of rapamycin. Dissecting domain-specific functions of Shp2 using double and truncation mutants, we determined that the hypertrophic effects of mutant Shp2 depend on the two SH2 domains and on an intact catalytic center. The latter finding prompted us to test the efficacy of a Shp2 inhibitor targeted directly at the catalytic pocket. This compound, PHPS1, effectively prevented mutant Shp2-induced hypertrophy. In summary, we identified three novel targets for pharmacological therapy of LS-associated cardiac hypertrophy. Of particular importance is the finding that intervention directly at the mutant Shp2 protein is effective because this would facilitate custom-tailored therapeutic approaches for patients carrying LS mutations in Shp2.     

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.     

Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.     

PZR Coordinates Shp2 Noonan and LEOPARD Syndrome Signaling in Zebrafish and Mice

Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, which manifests in congenital heart disease, short stature, and facial dysmorphia. The complexity of Shp2 signaling is exemplified by the observation that LEOPARD syndrome (LS) patients possess inactivating PTPN11 mutations yet exhibit similar symptoms to NS. Here, we identify “protein zero-related” (PZR), a transmembrane glycoprotein that interfaces with the extracellular matrix to promote cell migration, as a major hyper-tyrosyl-phosphorylated protein in mouse and zebrafish models of NS and LS. PZR hyper-tyrosyl phosphorylation is facilitated in a phosphatase-independent manner by enhanced Src recruitment to NS and LS Shp2. In zebrafish, PZR overexpression recapitulated NS and LS phenotypes. PZR was required for zebrafish gastrulation in a manner dependent upon PZR tyrosyl phosphorylation. Hence, we identify PZR as an NS and LS target. Enhanced PZR-mediated membrane recruitment of Shp2 serves as a common mechanism to direct overlapping pathophysiological characteristics of these PTPN11 mutations.     

Disease-causing SAP mutants are defective in ligand binding and protein folding

The X-linked lymphoproliferative (XLP) syndrome is caused by mutations or deletions in the SH2D1A gene that encodes an SH2 domain protein named SH2D1A or SAP. The identification of a number of missense mutations within the protein's SH2 domain, each of which can directly cause disease, provides a unique opportunity to investigate the function of an interaction protein module, SH2, in the pathogenesis of XLP. We show here that SAP mutants found in XLP patients are defective in binding its physiological ligands signaling lymphocyte activating molecule (SLAM), a co-receptor in T cell activation, and Fyn, a Src family protein tyrosine kinase. Consequently, these mutants are deficient in signaling through the SLAM receptor. This is reflected by compromised abilities for the mutants to recruit Fyn to SLAM and to activate Fyn, by reduced phosphorylation of the receptor, and by deficiencies for the mutants in blocking binding of SHP-2 to SLAM. Furthermore, all mutants examined are defective in protein folding as manifested by their significantly reduced melting temperatures upon thermal denaturation, compared to that of SAP. Taken together, these results suggest that defects in ligand binding, receptor signaling, and protein folding collectively contribute to the loss of function for disease-causing SAP mutants.     

Deletion of Shp2 Tyrosine Phosphatase in Muscle Leads to Dilated Cardiomyopathy,Insulin Resistance,and Premature Death

The intracellular signaling mechanisms underlying the pathogenesis of cardiac diseases are not fully understood. We report here that selective deletion of Shp2, an SH2-containing cytoplasmic tyrosine phosphatase, in striated muscle results in severe dilated cardiomyopathy in mice, leading to heart failure and premature mortality. Development of cardiomyopathy in this mouse model is coupled with insulin resistance, glucose intolerance, and impaired glucose uptake in striated muscle cells. Shp2 deficiency leads to upregulation of leukemia inhibitory factor-stimulated phosphatidylinositol 3-kinase/Akt, Erk5, and Stat3 pathways in cardiomyocytes. Insulin resistance and impaired glucose uptake in Shp2-deficient mice are at least in part due to impaired protein kinase C-ζ/λ and AMP-kinase activities in striated muscle. Thus, we have generated a mouse line modeling human patients suffering from cardiomyopathy and insulin resistance. This study reinforces a concept that a compound disease with multiple cardiovascular and metabolic disturbances can be caused by a defect in a single molecule such as Shp2, which modulates multiple signaling pathways initiated by cytokines and hormones.Heart failure is a serious life-threatening health problem worldwide. Numerous studies have demonstrated a link between cardiac dysfunction and insulin resistance, as well as deficiency in glucose transport (9, 35, 48). In the absence of manifest diabetes, insulin resistance and minor degrees of glucose intolerance are thought to be associated with and contribute to the development of nonischemic cardiomyopathy or idiopathic dilated cardiomyopathy (35, 45). However, the molecular basis for this link is poorly understood.Muscle-specific gene knockout mice have presented unprecedented opportunities to decipher molecular signaling mechanisms underlying cardiomyopathic changes. Deletion of PTEN in cardiomyocytes mediated by Mck-Cre results in cardiac hypertrophy in mice (8). Dilated cardiomyopathy was also observed to various degrees in mice with conditional ablation of ErbB2 (HER2), β₁ integrin, and the gp130 cytokine receptor component in the heart or muscle (16, 34, 37). Interestingly, despite the development of cardiomyopathy, most of these mutant mice survive to adulthood with a normal life span, suggesting limitations in their modeling of human patients'' pathological processes. These mutant mouse models also show no correlation between cardiomyopathy and insulin resistance. In fact, although muscle-specific PTEN knockout mice develop cardiac hypertrophy (8), they are protected against insulin resistance and diabetes induced by high-fat diet due to enhanced insulin-stimulated glucose uptake in soleus muscle (43).Shp2 is a widely expressed cytoplasmic tyrosine phosphatase with two SH2 domains that has been implicated in signaling events downstream of receptors for growth factors, cytokines, and hormones (25, 32). In particular, Shp2 has been shown to participate in leptin and insulin signaling for the regulation of energy balance and metabolism (23, 28, 46). In recent experiments, several groups have identified germ line gain and loss-of-function mutations in the human gene PTPN11, encoding Shp2, in Noonan syndrome and LEOPARD (for lentigines, electrocardiogram abnormalities, ocular hypertelorism, pulmonic valvular stenosis, abnormalities of genitalia, retardation of growth, and deafness) syndrome patients, respectively (21, 42). Paradoxically, these mutations either constitutively activate or inactivate the phosphatase activity leading to heart diseases, among other disorders observed in Noonan or LEOPARD syndrome patients. Since the conventional Shp2 knockout mice are embryonic lethal (36), tissue-specific deletion of Shp2 will be required to determine a specific function for Shp2 in the cardiovascular system in vivo.We report here that striated muscle-specific Shp2 knockout (MSKO) mice develop a severe dilated cardiomyopathy, resulting in heart failure and premature death in mice. More importantly, development of cardiomyopathy is associated with insulin resistance, glucose intolerance, and impaired insulin-stimulated glucose uptake in striated muscle cells in this mouse model.     

Counteracting Effects Operating on Src Homology 2 Domain-containing Protein-tyrosine Phosphatase 2 (SHP2) Function Drive Selection of the Recurrent Y62D and Y63C Substitutions in Noonan Syndrome

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.     

Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some,but not all,growth factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.     

Genome wide analysis of pathogenic SH2 domain mutations

The authors have made a genome-wide analysis of mutations in Src homology 2 (SH2) domains associated with human disease. Disease-causing mutations have been detected in the SH2 domains of cytoplasmic signaling proteins Bruton tyrosine kinase (BTK), SH2D1A, Ras GTPase activating protein (RasGAP), ZAP-70, SHP-2, STAT1, STAT5B, and the p85alpha subunit of the PIP3. Mutations in the BTK, SH2D1A, ZAP70, STAT1, and STAT5B genes have been shown to cause diverse immunodeficiencies, whereas the mutations in RASA1 and PIK3R1 genes lead to basal carcinoma and diabetes, respectively. PTPN11 mutations cause Noonan sydrome and different types of cancer, depending mainly on whether the mutation is inherited or sporadic. We collected and analyzed all known pathogenic mutations affecting human SH2 domains by bioinformatics methods. Among the investigated protein properties are sequence conservation and covariance, structural stability, side chain rotamers, packing effects, surface electrostatics, hydrogen bond formation, accessible surface area, salt bridges, and residue contacts. The majority of the mutations affect positions essential for phosphotyrosine ligand binding and specificity. The structural basis of the SH2 domain diseases was elucidated based on the bioinformatic analysis.     

The PTPN11 loss-of-function mutation Q510E-Shp2 causes hypertrophic cardiomyopathy by dysregulating mTOR signaling

The identification of mutations in PTPN11 (encoding the protein tyrosine phosphatase Shp2) in families with congenital heart disease has facilitated mechanistic studies of various cardiovascular defects. However, the roles of normal and mutant Shp2 in the developing heart are still poorly understood. Furthermore, it remains unclear how Shp2 loss-of-function (LOF) mutations cause LEOPARD Syndrome (also termed Noonan Syndrome with multiple lentigines), which is characterized by congenital heart defects such as pulmonary valve stenosis and hypertrophic cardiomyopathy (HCM). In normal hearts, Shp2 controls cardiomyocyte size by regulating signaling through protein kinase B (Akt) and mammalian target of rapamycin (mTOR). We hypothesized that Shp2 LOF mutations dysregulate this pathway, resulting in HCM. For our studies, we chose the Shp2 mutation Q510E, a dominant-negative LOF mutation associated with severe early onset HCM. Newborn mice with cardiomyocyte-specific overexpression of Q510E-Shp2 starting before birth displayed increased cardiomyocyte sizes, heart-to-body weight ratios, interventricular septum thickness, and cardiomyocyte disarray. In 3-mo-old hearts, interstitial fibrosis was detected. Echocardiographically, ventricular walls were thickened and contractile function was depressed. In ventricular tissue samples, signaling through Akt/mTOR was hyperactivated, indicating that the presence of Q510E-Shp2 led to upregulation of this pathway. Importantly, rapamycin treatment started shortly after birth rescued the Q510E-Shp2-induced phenotype in vivo. If rapamycin was started at 6 wk of age, HCM was also ameliorated. We also generated a second mouse model in which cardiomyocyte-specific Q510E-Shp2 overexpression started after birth. In contrast to the first model, these mice did not develop HCM. In summary, our studies establish a role for mTOR signaling in HCM caused by Q510E-Shp2. Q510E-Shp2 overexpression in the cardiomyocyte population alone was sufficient to induce the phenotype. Furthermore, the pathomechanism was triggered pre- but not postnatally. However, postnatal rapamycin treatment could still reverse already established HCM, which may have important therapeutic implications.     

Structural and functional effects of disease-causing amino acid substitutions affecting residues Ala72 and Glu76 of the protein tyrosine phosphatase SHP-2

Mutations of the protein tyrosine phosphatase SHP-2 are implicated in human diseases, causing Noonan syndrome (NS) and related developmental disorders or contributing to leukemogenesis depending on the specific amino acid substitution involved. SHP-2 is composed by a catalytic (PTP) and two regulatory (N-SH2 and C-SH2) domains that bind to signaling partners and control the enzymatic activity by limiting the accessibility of the catalytic site. Wild type SHP-2 and four disease-associated mutants recurring in hematologic malignancies (Glu76Lys and Ala72Val) or causing NS (Glu76Asp and Ala72Ser), with affected residues located in the PTP-interacting region of the N-SH2 domain, were analyzed by molecular dynamics simulations and in vitro biochemical assays. Simulations demonstrate that mutations do not affect significantly the conformation of the N-SH2 domain. Rather they destabilize the interaction of this domain with the catalytic site, with more evident effects in the two leukemia associated mutants. Consistent with this structural evidence, mutants exhibit an increased level of basal phosphatase activity in the order Glu76Lys > Ala72Val > Glu76Asp > Ala72Ser > WT. The experimental data also show that the mutants with higher basal activity are more responsive to an activating phosphopeptide. A thermodynamic analysis demonstrates that an increase in the overall phosphopeptide affinity of mutants can be explained by a shift in the equilibrium between the inactive and active SHP-2 structure. These data support the view that an increase in the affinity of SHP-2 for its binding partners, caused by destabilization of the closed, inactive conformation, rather than protein basal activation per se, would represent the molecular mechanism, leading to pathogenesis in these mutants.     

Phosphoproteomics-Mediated Identification of Fer Kinase as a Target of Mutant Shp2 in Noonan and LEOPARD Syndrome

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.     

SHP-2 acts via ROCK to regulate the cardiac actin cytoskeleton

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.     

Noonan syndrome-associated SHP-2/Ptpn11 mutants enhance SIRPalpha and PZR tyrosyl phosphorylation and promote adhesion-mediated ERK activation

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.     

A weak Lck tail bite is necessary for Lck function in T cell antigen receptor signaling

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH) 2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.