C-SH2 Domain Research Articles

Engineering the Recruitment of Phosphotyrosine Binding Domain-containing Adaptor Proteins Reveals Distinct Roles for RET Receptor-mediated Cell Survival

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.

Reduction of p66Shc suppresses oxidative damage in retinal pigmented epithelial cells and retina

The largest isoform of the Shc adapter protein, p66Shc, has been implicated in oxidative damage-induced apoptosis in vital organs, because mice deficient in p66Shc have a 30% increase in life span and are resistant to the lethal effects of systemically administered paraquat, a source of severe oxidative damage. In this study, we utilized siRNA directed against the CH2 domain of Shc, to reduce p66Shc, but not p52Shc nor p46Shc in retinal pigmented epithelial (RPE) cells. RPE cells deficient in p66Shc had reduced susceptibility to oxidative stress-induced apoptosis. Compared to control cells, those with reduced p66Shc had increased basal and oxidative stress-induced NF-kappaB transcriptional activity, increased levels of antioxidant enzymes, and less generation of reactive oxygen species when challenged with H(2)O(2). The increase in oxidative stress-induced NF-kappaB activity was mediated by activation of ERK. Compared to eyes injected with GFP siRNA, those injected with p66Shc siRNA showed less loss of retinal function as assessed by electroretinograms from paraquat-induced oxidative stress. These data suggest that p66Shc and molecular signals involved in its regulation provide therapeutic targets for retinal degenerations in which oxidative-damage plays a major role, including age-related macular degeneration and cone cell death in retinitis pigmentosa.

Sequence Specificity of SHP-1 and SHP-2 Src Homology 2 Domains: CRITICAL ROLES OF RESIDUES BEYOND THE pY+3 POSITION

A combinatorial phosphotyrosyl (pY) peptide library was screened to determine the amino acid preferences at the pY+4 to pY+6 positions for the four SH2 domains of protein-tyrosine phosphatases SHP-1 and SHP-2. Individual binding sequences selected from the library were resynthesized and their binding affinities and specificities to various SH2 domains were further evaluated by SPR studies, stimulation of SHP-1 and SHP-2 phosphatase activity, and in vitro pulldown assays. These studies reveal that binding of a pY peptide to the N-SH2 domain of SHP-2 is greatly enhanced by a large hydrophobic residue (Trp, Tyr, Met, or Phe) at the pY+4 and/or pY+5 positions, whereas binding to SHP-1 N-SH2 domain is enhanced by either hydrophobic or positively charged residues (Arg, Lys, or His) at these positions. Similar residues at the pY+4 to pY+6 positions are also preferred by SHP-1 and SHP-2 C-SH2 domains, although their influence on the overall binding affinities is much smaller compared with the N-SH2 domains. A structural model was generated to qualitatively interpret the contribution of the pY+4 and pY+5 residues to the overall binding affinity. Examination of pY motifs from known SHP-1 and SHP-2-binding proteins shows that many of the pY motifs contain a hydrophobic or positively charged residue(s) at the pY+4 and pY+5 positions.

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II.

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized. Several studies suggest that the C-terminal SH3 domain plays an important regulatory role in the protein; however, no structural information is available on this domain, and relatively little is known about its binding partners. In the current work, we have solved the solution NMR structure of the C-terminal SH3 domain. The domain adopts the standard SH3 fold comprising a five-stranded beta barrel. In agreement with alignment and modeling studies, the structure indicates that the canonical-binding surface of the SH3 domain is unusually polar and suggests that this domain may not bind typical PXXP ligands or that it may bind them with reduced affinity. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. These studies offer some insight into the likely structural and thermodynamic consequences of point mutations in the cSH3 domain that are known to deregulate Crk-II function. Our results set the stage for a better understanding the role of the cSH3 domain in the context of the full-length protein.

An active form of Vav1 induces migration of mammary epithelial cells by stimulating secretion of an epidermal growth factor receptor ligand

BackgroundVav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.ResultsWe found that expression of an active form of Vav1, Vav1Y3F, in MCF-10A mammary epithelial cells increases cell migration in the absence or presence of EGF. Vav1Y3F was also able to drive Rac1 activation and PAK and ERK phosphorylation in MCF-10A cells in the absence of EGF stimulation. Mutations in the Dbl homology, pleckstrin homology, or cysteine-rich domains of Vav1Y3F abolished Rac1 or ERK activation in the absence of EGF and blocked the migration-promoting activity of Vav1Y3F. In contrast, mutations in the SH2 and C-SH3 domains did not affect Rac activation by Vav1Y3F, but reduced the ability of Vav1Y3F to induce EGF-independent migration and constitutive ERK phosphorylation. EGF-independent migration of MCF-10A cells expressing Vav1Y3F was abolished by treatment of cells with an antibody that prevents ligand binding to the EGF receptor. In addition, conditioned media collected from Vav1Y3F expressing cells stimulated migration of parental MCF-10A cells. Lastly, treatment of cells with the EGF receptor inhibitory antibody blocked the Vav1Y3F-induced, EGF-independent stimulation of ERK phosphorylation, but had no effect on Rac1 activation or PAK phosphorylation.ConclusionOur results indicate that increased migration of active Vav1 expressing cells is dependent on Vav1 GEF activity and secretion of an EGF receptor ligand. In addition, activation of ERK downstream of Vav1 is dependent on autocrine EGF receptor stimulation while active Vav1 can stimulate Rac1 and PAK activation independent of ligand binding to the EGF receptor. Thus, stimulation of migration by activated Vav1 involves both EGF receptor-dependent and independent activities induced through the Rho GEF domain of Vav1.

Regulation and Function of SKAP-55 Non-canonical Motif Binding to the SH3c Domain of Adhesion and Degranulation-promoting Adaptor Protein

The immune cell adaptor adhesion and degranulation promoting adaptor protein (ADAP) and its binding to T-cell adaptor Src kinase-associated protein of 55 kDa (SKAP-55) play a key role in the modulation of T-cell adhesion. While primary binding occurs via SKAP-55 SH3 domain binding to a proline-rich region in ADAP, a second interaction occurs between the ADAP C-terminal SH3 domain (ADAP-SH3c) and a non-canonical RKXXY294XXY297 motif in SKAP-55. Increasing numbers of non-canonical SH3 domain binding motifs have been identified in a number of biological systems. The presence of tyrosine residues in the SKAP-55 RKXXY294XXY297 motif suggested that phosphorylation might influence this unusual SH3 domain interaction. Here, we show that the Src kinase p59fyn can induce the in vivo phosphorylation of the motif, and this event blocks ADAP-SH3c domain binding to the peptide motif. The importance of tyrosine phosphorylation was confirmed by plasmon resonance interaction analysis showing that phosphorylation of Tyr294 residue plays a central role in mediating dissociation, whereas phosphorylation of the second Tyr297 had no effect. Although loss of this secondary interaction did not result in the disruption of the complex, the Y294F mutation blocked T-cell receptor-induced up-regulation of lymphocyte function-associated antigen-1-mediated adhesion to intercellular adhesion molecule-1 and interleukin-2 promoter activity. Our findings identify a RKXXY294 motif in SKAP-55 that mediates unique ADAP SH3c domain binding and is needed for LFA-1-mediated adhesion and cytokine production.

The Smaller Isoforms of Ankyrin 3 Bind to the p85 Subunit of Phosphatidylinositol 3′-Kinase and Enhance Platelet-derived Growth Factor Receptor Down-regulation

The Src homology 2 (SH2) domains of the p85 subunit of phosphatidylinositol 3'-kinase have been shown to bind to the tyrosine-phosphorylated platelet-derived growth factor receptor (PDGFR). Previously, we have demonstrated that p85 SH2 domains can also bind to the serine/threonine kinase A-Raf via a unique phosphorylation-independent interaction. In this report, we describe a new phosphotyrosine-independent p85 SH2-binding protein, ankyrin 3 (Ank3). In general, ankyrins serve a structural role by binding to both integral membrane proteins at the plasma membrane and spectrin/fodrin proteins of the cytoskeleton. However, smaller isoforms of Ank3 lack the membrane domain and are localized to late endosomes and lysosomes. We found that p85 binds directly to these smaller 120- and 105-kDa Ank3 isoforms. Both the spectrin domain and the regulatory domain of Ank3 are involved in binding to p85. At least two domains of p85 can bind to Ank3, and the interaction involving the p85 C-SH2 domain was found to be phosphotyrosine-independent. Overexpression of the 120- or 105-kDa Ank3 proteins resulted in significantly enhanced PDGFR degradation and a reduced ability to proliferate in response to PDGF. Ank3 overexpression also differentially regulated signaling pathways downstream from the PDGFR. Chloroquine, an inhibitor of lysosomal-mediated degradation pathways, blocked the ability of Ank3 to enhance PDGFR degradation. Immunofluorescence experiments demonstrated that both small Ank3 isoforms colocalized with the lysosomal-associated membrane protein and with p85 and the PDGFR. These results suggest that Ank3 plays an important role in lysosomal-mediated receptor down-regulation, likely through a p85-Ank3 interaction.

Structural and Signaling Requirements for Transformation Mediated by Shp2 Mutants.

The SH2-containing tyrosine phosphatase Shp2 (PTPN11) is required for normal growth factor and cytokine signaling. Germline Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, and at lower incidence in various chronic and acute leukemias. JMML patients without Shp2 mutations have either activating Ras mutations or homozygotic inactivation of Nf1. Nearly all disease-associated Shp2 mutations affect residues known to control catalytic activity. NS and leukemia mutations can involve the same residues, but when they do, the latter are less conservative, suggesting that they may be more activating. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Leukemia-associated mutants yielded much higher number and larger size of colonies than NS-associated mutants (e.g., N308D, N308S, E76D) whereas, mutants associated with both NS and leukemia (T73I, E139D, Q506P) evoked intermediate numbers. Transplantation of E76K− or D61Y− (but not parental virus or WT Shp2) transduced BM into lethally irradiated recipients evoked a fatal JMML-like disorder. To understand the structural requirements of Shp2 for transformation, we made a series of second site mutations in Shp2 E76K and expressed these mutants in murine BM. Mutation of the canonical arginine residue to methionine in the FLVRES motif in either N-SH2 or C-SH2 domain of Shp2 E76K ablated myeloid transforming ability. Mutation of the conserved arginine residue within the Shp2 E76K phosphatase domain signature motif to methionine, which diminishes phosphatase activity, also eliminated myeloid transformation. Tyrosine residues 542 and 580 in the C-terminus of Shp2 are known to undergo phosphorylation in response to many growth factors and required for Grb2 binding. Mutation of tyrosine 542 to phenylalanine markedly reduced transformation, whereas tyrosine 580 mutation alone had little effect. However, mutation of both tyrosines eliminated Shp2 E76K-evoked transformation, suggesting that Grb2-binding to Shp2 could be important for transformation. We observed that Gab2, a major Shp2 SH2 domain binding protein, was absolutely required for transformation by Shp2 E76K but not activating RasV12. Gab3, another member of Gab family protein mainly expressed in hematopoietic cells, was not critical for Shp2 E76K-evoked transformation. We also found that Stat5 was important for Shp2 E76K-mediated transformation. Mast cells derived from E76K− and D61Y− transplanted mice BM exhibited increased proliferation and enhanced activation of Erk, p38MAPK, Akt and Stat5 in response to IL-3. These results provide some mechanistic insights into how Shp2 mutants could transform primary myeloid cells and result in JMML-like MPD.

Lck couples Shc to TCR signaling

Recent genetic evidence demonstrated that Shc is a critical molecule for T cell activation and differentiation. However, how Shc is coupled to the T cell antigen receptor (TCR) has not been clearly characterized. Here we report that the tyrosine kinase Lck functions as a connecting molecule for TCR and Shc. Lck plays a critical role in TCR signal transduction by phosphorylating the immuno-receptor tyrosine based activation motif (ITAM). Our data shows that the PTB domain of Shc binds the SH2/3 domains of Lck in a phosphotyrosine-independent manner. Inhibition of the Lck/Shc interaction led to the loss of IL-2 promoter activation, confirming that the role of Shc in IL-2 production requires its interaction with Lck. Together, the data show that Shc is connected to the activated TCR via direct interaction with Lck.

PI3-kinase activation by GM-CSF in endothelium is upstream of Jak/Stat pathway: Role of αGMR

GM-CSF has been identified as a growth factor for endothelial cells. In this study, we investigated the role of PI3-kinase pathway in mediating GM-CSF induced angiogenesis. GM-CSF induced tube formation in human umbilical vein endothelial cells, as examined using Matrigel assay, was inhibited by specific inhibitors of PI3-kinase, wortmannin, and LY294002. The regulatory subunit of PI3-kinase (p85) interacted with αGMR via its C-SH2 domain in a GM-CSF-dependent fashion with concomitant phosphorylation of p85 and activation of PI3-kinase pathway. p85 binding site on the αGMR was essential to induce GM-CSF receptor-dependent Stat activation. Furthermore, inhibition of PI3-kinase activity also abrogated GM-CSF induced Stat activation. These studies underscore the significance of the GM-CSF mediated PI3-kinase activation and its role in angiogenesis.

Mechanism of Constitutive Phosphoinositide 3-Kinase Activation by Oncogenic Mutants of the p85 Regulatory Subunit

p85/p110 phosphoinositide 3-kinases regulate multiple cell functions and are frequently mutated in human cancer. The p85 regulatory subunit stabilizes and inhibits the p110 catalytic subunit. The minimal fragment of p85 capable of regulating p110 is the N-terminal SH2 domain linked to the coiled-coil iSH2 domain (referred to as p85ni). We have previously proposed that the conformationally rigid iSH2 domain tethers p110 to p85, facilitating regulatory interactions between p110 and the p85 nSH2 domain. In an oncogenic mutant of murine p85, truncation at residue 571 leads to constitutively increased phosphoinositide 3-kinase activity, which has been proposed to result from either loss of an inhibitory Ser-608 autophosphorylation site or altered interactions with cellular regulatory factors. We have examined this mutant (referred to as p65) in vitro and find that p65 binds but does not inhibit p110, leading to constitutive p110 activity. This activated phenotype is observed with recombinant proteins in the absence of cellular factors. Importantly, this effect is also produced by truncating p85ni at residue 571. Thus, the phenotype is not because of loss of the Ser-608 inhibitory autophosphorylation site, which is not present in p85ni. To determine the structural basis for the phenotype of p65, we used a broadly applicable spin label/NMR approach to define the positioning of the nSH2 domain relative to the iSH2 domain. We found that one face of the nSH2 domain packs against the 581-593 region of the iSH2 domain. The loss of this interaction in the truncated p65 would remove the orienting constraints on the nSH2 domain, leading to a loss of p110 regulation by the nSH2. Based on these findings, we propose a general model for oncogenic mutants of p85 and p110 in which disruption of nSH2-p110 regulatory contacts leads to constitutive p110 activity.

Src homology domains in phospholipase C‐γ1 mediate its anti‐apoptotic action through regulating the enzymatic activity

Phospholipase-gamma1 (PLC-gamma1) prevents programmed cell death, for which the enzymatic activity has been implicated. However, the biological function of Src homology (SH) domains of PLC-gamma1 in promoting cell survival remains elusive. Here, we showed that deletion of the N-SH2 domain or both N-SH2 and C-SH2 domains, but not the SH3 domain, abolished the anti-apoptotic activity of PLC-gamma1. Surprisingly, removal of the whole SH domain inhibited apoptosis. The lipase-inactive PLC-gamma1 mutant (LIM) failed to suppress apoptosis. Moreover, the phospholipase activity in SH3- or whole SH domain-deleted cells was comparable to that of wild-type cells. By contrast, the enzymatic activity was substantially ablated in SH2 domain-deleted or LIM cells. A pharmacological inhibitor of PLC-gamma1 robustly diminished the anti-apoptotic action in wild-type, SH3- or whole SH domain-deleted cells, whereas pretreatment of SH2 domain-deleted or LIM cells with agents activating PKC and calcium mobilization markedly promoted cell survival. These results indicate that SH domains in PLC-gamma1 might mediate its anti-apoptotic action by regulating the enzymatic activity.

Intra- and intermolecular interactions mediated by adaptor protein Ruk/CIN85/SETA

Ruk/CIN85l SETA is a member of a separate and evolutionary conserved family of adapter I scaffold proteins implicated in apoptic and receptor tyrosine kinases signalling, rearrangement of actin cytoskeleton and cell adhesion, podocyte and T cell functions. Self-regulation through intra- and intercellular interactions can be supposed for RuklCIN85l SETA as this protein contains SH3 domains and proline-rich sequence, localized within one polypeptide chain, as well as C-terminal coiled-coil region. The ability of Ruk proline-rich motifs to interact with its own SH3 domains in an intramolecular fashion and coiled-coil region to mediate oligomerization between different isoforms was assessed in GST pull down experiments. It was shown that both Ruk SH3A and to a less extent SH3B domains can interact with its own proline-rich sequences in a cooperative manner, while coiled-coil region provide for isoforms oligomerization. SH3C domain appear exerts conformational constraints, imposed on coiled-coil region, restricting the level of oligomerization. We also demonstrated that the ability of exogenous ligands to interact with Ruk polyprotine motifs is changing during the course of TNFa-induced apoptosis of human myelomonocytic W37 cells.

Genotypic and phenotypic characterization of Noonan syndrome: New data and review of the literature

Noonan syndrome (NS) is an autosomal dominant disorder, characterized by short stature, minor facial anomalies, and congenital heart defects. In approximately 50% of cases the condition is caused by missense mutations in the PTPN11 gene on chromosome 12, resulting in a gain of function of the protein SHP-2. In this study, PTPN11 mutation analysis was performed in 170 NS patients. In 76 (45%) of them a mutation was identified. We report on the distribution of these mutations, as well as on genotype-phenotype relationships. The benefit of the NS scoring system developed by van der Burgt et al. [(1994); Am J Med Genet 53:187-191] is shown, among physicians who consequently based their diagnosis on the NS scoring system the percentage mutation positive subjects was 54%, whereas this percentage was only 39% among physicians who made less use of the scoring system. In two patients with some uncommon manifestations mutations were found in the C-SH2 domain, a region in which defects are not often identified in NS. A trend was observed in patients carrying the 922A --> G change (Asn308Asp) receiving normal education. In one patient with NS and mild juvenile myelomonocytic leukemia (JMML) the mutation 218C --> T (Thr73Ile) was found. This confirms previous findings indicating that individuals with NS with specific mutations in PTPN11 are at risk of developing JMML.

Novel Mechanism of Interaction of p85 Subunit of Phosphatidylinositol 3-Kinase and ErbB3 Receptor-derived Phosphotyrosyl Peptides

Ligand-activated and tyrosine-phosphorylated ErbB3 receptor binds to the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase and initiates intracellular signaling. Here, we studied the interactions between the N- (N-SH2) and C- (C-SH2) terminal SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase and eight ErbB3 receptor-derived phosphotyrosyl peptides (P-peptides) by using molecular dynamics, free energy, and surface plasmon resonance (SPR) analyses. In SPR analysis, these P-peptides showed no binding to the C-SH2 domain, but P-peptides containing a phospho-YXXM or a non-phospho-YXXM motif did bind to the N-SH2 domain. The N-SH2 domain has two phosphotyrosine binding sites in its N- (N1) and C- (N2) terminal regions. Interestingly, we found that P-peptides of pY1180 and pY1241 favored to bind to the N2 site, although all other P-peptides showed favorable binding to the N1 site. Remarkably, two phosphotyrosines, pY1178 and pY1243, which are just 63 amino acids apart from the pY1241 and pY1180, respectively, showed favorable binding to the N1 site. These findings indicate a possibility that the pair of phosphotyrosines, pY1178-pY1241 or pY1243-pY1180, will fold into an appropriate configuration for binding to the N1 and N2 sites simultaneously. Our model structures of the cytoplasmic C-terminal domain of ErbB3 receptor also strongly supported the speculation. The calculated binding free energies between the N-SH2 domain and P-peptides showed excellent qualitative agreement with SPR data with a correlation coefficient of 0.91. The total electrostatic solvation energy between the N-SH2 domain and P-peptide was the dominant factor for its binding affinity.

Analysis of a p67phoxΔ401 Mutant Identified in Chronic Granulomatous Disease: Deletion of C-Terminal SH3 Binding Site for p47phox Still Supports NADPH Oxidase Activity in Cosphox Cells.

The phagocyte NADPH oxidase plays an essential role in host defense through the production of reactive oxygen species. Oxidase activation occurs via assembly of cytosolic p47phox, p67phox, p40phox and Rac with the membrane flavocytochrome b558. This work presents the functional analysis of a mutant p67phox identified in two unrelated patients affected with chronic granulomatous disease (CGD), a 5 nucleotide deletion in exon 13 that generates a premature stop codon at amino acid position 401. The p67phox-Δ401 (p67-d401) protein, which lacks the wild type (WT) C-terminal residues 398–526, has a truncated PB1 domain while the C-SH3 domain is absent. In neutrophils and EBV-B lymphocytes derived from a patient with this mutant, the protein was not detected and RNA appears to be unstable (Patino et al. 1999). This mutant was transiently expressed in COS7 cells that also expressed gp91phox, p22phox, and p47phox (COS91-22-47 cells) or the first two of these (COS91-22), from stable transgenes. The p67-d401 protein was expressed at lower levels (~38%) compared to p67- WT when the same amount of plasmid DNA was used for transfection, which was consistently observed over a three fold range of plasmid DNA; this suggests instability of the mRNA or protein. When normalized for protein expression, NADPH oxidase activity in cells expressing p67-d401 was similar to those expressing p67-WT in response to PMA and 3 times better in response to arachidonic acid (AA). Co-expression of p47phox was necessary for p67-d401 to support O2- production in response to AA and PMA. Co-expression of p40phox did not affect either expression levels of p67-WT or p67-d401 or NADPH oxidase activity. Very little p67-WT was detected in membranes of transfected resting COS91-22-47 cells, with some membrane translocation observed following stimulation with PMA or AA, as expected. Interestingly, a significant amount of p67-d401 was detected in membranes and very little in the cytosol of resting COS91-22-47 cells transfected with this mutant, similar to C-terminal truncation mutants studied by de Mendez and colleagues (de Mendez et al 1994, de Mendez et al 1996). Upon activation with AA or PMA, a ~1.5 fold increase in p67-d401 and a ~ 1.6 fold increase in p47phox associated with the membrane fraction was observed in COS91-22-47 cells transfected with p67-d401. Finally, transiently transfected p67-d401 co-immunoprecipitated with p47phox both in resting and stimulated COS91-22-47 cells. This indicates that the carboxyl terminal SH3 domain of p67phox is not the only site of interaction between p67phox and p47phox, and suggests that a second binding site exists. In conclusion, the COSphox system represents a transfectable whole cell model for functional analysis of mutant phox proteins with reduced stability in neutrophils. It is likely that over expression of the p67-d401 cDNA in this system permits detectable expression of recombinant p67-d401. The unusual properties of the p67-d401 mutant, with spontaneous membrane translocation as well as ability to interact with p47phox in the absence of the C-terminal p67phox SH3 domain may provide further insight into the multiplicity of interactions between each of the oxidase subunits and their relation with the Rac and the cytochrome b558.

Phosphatidylinositol 3-Kinase p85 Subunit-Dependent Interaction with BCR/ABL: Molecular Mechanism and Biological Consequences.

BCR/ABL fusion tyrosine kinase is responsible for the initiation and maintenance of the Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) and a cohort of acute lymphocytic leukemias (ALL). Our previous studies showed that a signaling protein phosphatidylinositol-3 kinase (PI-3k) is essential for the growth of CML cells, but not of normal hematopoietic cells, and that p85 subunit of PI-3k co-immunoprecipitates with BCR/ABL (Skorski et al., (1995) Blood 86, 726–36; Skorski et al., (1997) Embo J 16, 6151–61; Klejman et al., (2002) Oncogene 21, 5868–76). Therefore, we made an attempt to better characterize the p85 - BCR/ABL interactions. Mutagenesis approach combined with pull-down assays indicated that both N-terminal and C-terminal SH2 domains (nSH2 and cSH2, respectively) and SH3 domain of p85 play an important role in the interaction with BCR/ABL. SH2 domains exerted their function through binding to the tyrosine-phosphorylated motifs, and SH3 domain recognized proline-rich regions. Disruption of these functions by introduction of point-mutations in nSH2, cSH2 and SH3 domains abrogated their interaction with BCR/ABL. p85 mutant protein (p85-mut) bearing these mutations was not able to interact with BCR/ABL, while its binding to the p110 catalytic subunit of PI-3k was intact. In addition, binding of Shc and Gab2, but not Crk-L, to p85-mut was abrogated. When expressed in BCR/ABL-transformed hematopoietic cells p85-mut diminished activation of Akt kinase, the downstream effector of PI-3k. This effect was associated with the inhibition of BCR/ABL-dependent growth of hematopoeitic cells line and murine bone marrow cells. Interestingly, addition of IL-3 rescued BCR/ABL-transformed cells from the inhibitory effect of p85-mut. SCID mice injected with BCR/ABL-positive hematopoietic cells expressing p85-mut survived longer in comparison to the animals inoculated with BCR/ABL-transformed counterparts transfected with empty plasmid. In conclusion, we have identified the domains of p85 responsible for the interaction with BCR/ABL. Moreover, we demonstrated that expression of p85-mut, which binds to the p110 catalytic subunit of PI-3k but is not able to interact with BCR/ABL, affected the growth ability of BCR/ABL-positive leukemia cells.

The iSH2 domain of PI 3-kinase is a rigid tether for p110 and not a conformational switch

Class IA PI 3-kinases are heterodimeric proteins with distinct catalytic (p110) and regulatory (p85) subunits. The minimal fragment of p85 capable of regulating p110 activity (p85ni) is the N-terminal SH2 domain linked to the iSH2 coiled-coil domain. We used cysteine mutagenesis and 14C-NEM-labeling to show that the p110-binding site in the iSH2 domain includes two regions: residues 482–484 and 532–541. These regions are adjacent to each other in the three-dimensional structural model of the iSH2 domain, and define a coherent binding site. We then used spin labeling and EPR spectroscopy to demonstrate that the conformation of the iSH2 domain is unaffected by binding to the N-terminal fragment of p110 (residues 1–108), and/or by phosphopeptide binding to p85ni/p110(1–108) heterodimers. Finally, we show that the cSH2 domain cannot substitute for the nSH2 domain with regard to inhibition of p110. These data support a model in which the iSH2 domain is a rigid tether for p110, and regulation of p85/p110 is mediated by nSH2-p110 contacts.

Genetics and Variation in Phenotype in Noonan Syndrome

Noonan syndrome is a well-known clinical entity comprising multiple congenital anomalies characterized by typical facial features, short stature and congenital heart defect. Approximately 50% of cases are sporadic. Familial cases are generally autosomal dominant. In 2001 a gene responsible for Noonan syndrome, PTPN11, encoding for the non-receptor protein tyrosine phosphatase SHP-2, was identified. Mutation analysis of the PTPN11 gene was carried out in Nijmegen in 150 patients with Noonan syndrome. Mutations were found in 68 patients (45%), the most common being A922G in exon 8. In exon 4 a mutation was found that encoded the C-SH2 domain of the PTPN11 gene in two unique patients who shared some uncommon features. A 218C→T mutation was found in exon 3 in one patient with Noonan syndrome and mild juvenile myelomonocytic leukaemia.

Alternative splicing of mammalian Intersectin 1: domain associations and tissue specificities

The Intersectin 1 (ITSN1) protein functions in clathrin-mediated endocytosis and in MAP kinase signaling. The complex domain structure comprises two EH and five SH3 domains in the short isoform, plus RhoGEF, pleckstrin, and putative calcium-interaction domains in the long isoform. Alternative splicing of exon 20, affecting the SH3A domain, has been shown in rat and that of exons 25 + 26, affecting the SH3C domain, has been shown in human and rat. Here we report 7 novel splice variants of the human and mouse ITSN1 genes and demonstrate conservation of alternative splicing affecting SH3A and SH3C in mouse. The novel variants encode transcripts with altered EH domain spacing and RhoGEF domain structure and possible targets of nonsense-mediated decay. Eight and 16 protein variants of the short and long ITSN1 isoforms, respectively, are predicted. These isoforms likely serve to modulate the many complex protein interactions and functions of ITSN1.