Uncoupling Crk signal transduction by Pseudomonas exoenzyme T

Exoenzyme T (ExoT) is a bifunctional type III cytotoxin of Pseudomonas aeruginosa that possesses both Rho GTPase-activating protein and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity of ExoT stimulated depolymerization of the actin cytoskeleton independent of Rho GTPase-activating protein function, and ExoT was subsequently shown to ADP-ribosylate Crk (CT10 regulator of kinase)-I and Crk-II. Crk proteins are eukaryotic adaptor proteins comprising SH2 and SH3 domains that are components of the integrin signaling pathway leading to Rac1 and Rap1 functions. Mass spectroscopic analysis identified Arg20 as the site of ADP-ribosylation by ExoT. Arg20 is a conserved residue located within the SH2 domain that is required for interactions with upstream signaling molecules such as paxillin and p130cas. Glutathione S-transferase pull-down and far Western assays showed that ADP-ribosylated Crk-I or Crk-I(R20K) failed to bind p130cas or paxillin. This indicates that ADP-ribosylation inhibited the direct interaction of Crk with these focal adhesion proteins. Overexpression of wild-type Crk-I reduced cell rounding by ExoT, whereas expression of dominant-active Rac1 interfered with the ability of ExoT to round cells. Thus, the ADP-ribosylation of Crk uncouples integrin signaling by direct inhibition of the binding of Crk to focal adhesion proteins.     

Tyrosine phosphorylated Disabled 1 recruits Crk family adapter proteins

Disabled 1 (Dab1) functions as a critical adapter protein in the Reelin signaling pathway to direct proper positioning of neurons during brain development. Reelin stimulates phosphorylation of Dab1 on tyrosines 198 and 220, and phosphorylated Dab1 is likely to interact with downstream signaling proteins that contain Src homology 2 (SH2) domains. To search for such proteins, we used a Sepharose-conjugated peptide containing phosphotyrosine 220 (PTyr-220) of Dab1, as an affinity matrix to capture binding proteins from mouse brain extracts. Mass spectrometric analysis of bound proteins revealed that Crk family adapter proteins selectively associated with this phosphorylation site. We further show that Crk-I and Crk-II, but not CrkL, stimulate phosphorylation of Dab1 on tyrosine 220 in a Src-dependent manner. Our results suggest that Crk family adapter proteins may play an important role in the Reelin signaling pathway during brain development.     

Mutational analysis of the interaction between insulin receptor and IGF-I receptor with c-Crk and Crk-L in a yeast two-hybrid system

The SH2/SH3 adapter proteins of the Crk family are potent signal transducers after receptor tyrosine kinase stimulation with insulin or IGF-1. We have employed a yeast two-hybrid approach and mutational analysis to dissect the capabilities of the insulin receptor and the IGF-I receptor to directly associate with Crk isoforms. Insulin receptor stably recruits full length Crk by association with its SH2 domain in an auto-phosphorylation dependent manner. In contrast, interaction of the IGF-I receptor with the Crk-IISH2 domain was only detectable when Crk-II was truncated in its C-terminal part, indicating the transient nature of this interaction. From these data it can be concluded that members of the insulin receptor family activate Crk proteins in a differential manner.     

Impaired expression of importin/karyopherin beta1 leads to post-implantation lethality

Importin beta1 (Impbeta)/karyopherin beta1 (Kpnb1) mediates the nuclear import of a large variety of substrates. This study aimed to investigate the requirement for the Kpnb1 gene in mouse development, using a gene trap line, B6-CB-Ayu8108(GtgeoIMEG) (Ayu8108(geo)), in which the trap vector was inserted into the promoter region of the Kpnb1 gene, but in reverse orientation of the Kpnb1 gene. Ayu8108(geo/geo) homozygous embryos could develop to the blastocyst stage, but died before embryonic day 5.5, and expression of the Kpnb1 gene in homozygous blastocysts was undetectable. We also replaced the betageo gene with Impbeta cDNA through Cre-mediated recombination to rescue Impbeta expression. Homozygous mice for the rescued allele Ayu8108(Impbeta/Impbeta) were born and developed normally. These results demonstrated that the cause of post-implantation lethality of Ayu8108(geo/geo) homozygous embryos was impaired expression of the Kpnb1 gene, indicating indispensable roles of Impbeta1 in early development of mice.     

Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin

The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.     

Characterization of Ayu17-449 gene expression and resultant kidney pathology in a knockout mouse model

The gene trap technique is a powerful approach for characterizing and mutating genes in the mouse. We used this method to identify a mouse gene of unknown function and to establish a mutant mouse line. We subsequently identified one gene, denoted Ayu17-449, on mouse chromosome 3 that comprised 14 exons encoding 1920 amino acids with a granin motif in its N-terminal sequence. In adult mice, this gene was highly expressed in the brain, heart, lung, muscle, stomach, and kidney. The insertion of a trap vector into the second intron of this gene resulted in the null mutation. Homozygous mice for these mutation died by 1 day after birth. Mutant mice showed a loss of acidic granules in the proximal convoluted tubules of the kidney. Our data demonstrates that Ayu17-449 is important for mouse survival.     

Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 regulator of kinase (Crk) proteins

Pseudomonas aeruginosa ExoT is a type III cytotoxin that functions as an anti-internalization factor with an N-terminal RhoGAP domain and a C-terminal ADP-ribosyltransferase domain. Although ExoT RhoGAP stimulates actin reorganization through the inactivation of Rho, Rac, and Cdc42, the function of the ADP-ribosylation domain is unknown. The present study characterized the mammalian proteins that are ADP-ribosylated by ExoT, using two-dimensional SDS-PAGE and matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) analysis. ExoT ADP-ribosylated two cytosolic proteins in cell lysates upon type III delivery into cultured HeLa cells. MALDI-TOF mass spectrometry analysis identified the two proteins as Crk-I and Crk-II that are Src homology 2-3 domains containing adaptor proteins, which mediate signal pathways involving focal adhesion and phagocytosis. ExoT ADP-ribosylated recombinant Crk-I at a rate similar to the ADP-ribosylation of soybean trypsin inhibitor by ExoS. ExoS did not ADP-ribosylate Crk-I. ADP-ribosylation of Crk-I may be responsible for the anti-phagocytosis phenotype elicited by ExoT in mammalian cells.     

The substrate specificity of the catalytic domain of Abl plays an important role in directing phosphorylation of the adaptor protein Crk

c-Abl preferentially phosphorylates peptide substrates that contain proline at the P+3 site (relative to the phosphorylated tyrosine). We previously described a mutant form of the Abl catalytic domain (Y569W) with altered substrate specificity at the P+3 position, as measured using synthetic peptides. In this study, we examine the phosphorylation of Crk, a protein substrate of Abl that is phosphorylated in the sequence Tyr221-Ala-Gln-Pro. In vitro, phosphorylation of Crk by Y569W Abl is greatly reduced relative to wild-type Abl. Overexpression of Y569W mutant Abl in 293T kidney cells produces a similar overall pattern of tyrosine phosphorylation as wild-type Abl, indicating that not all cellular proteins depend on Pro at P+3 for Abl recognition. However, phosphorylation of Crk by Y569W Abl in these cells is markedly reduced relative to wild-type Abl. A truncated form of Abl lacking the C-terminal polyproline region is not able to phosphorylate Crk in these assay conditions. Thus, proper phosphorylation of Crk by Abl depends not only on the interaction of the Crk SH3 domain with the Abl polyproline region, but also on the recognition of amino acids surrounding tyrosine by the Abl catalytic domain.     

Commentary: The carboxyl-terminal Crk SH3 domain: Regulatory strategies and new perspectives

Since their discovery as cellular counterparts of viral oncogenes more than two decades ago, enormous progress has been made in unraveling the complex regulatory pathways of signal transduction initiated by the Crk family of proteins. New structural and biochemical studies have uncovered novel insights into both negative and positive regulation of Crk mediated by its atypical carboxyl-terminal SH3 domain (SH3C). Moreover, SH3C is tyrosine phosphorylated by receptor tyrosine kinases and non-receptor tyrosine kinases, thereby permitting assemblages of other SH2/PTB domain containing proteins. Such non-canonical signaling by the Crk SH3C reveals new regulatory strategies for adaptor proteins.     

Fibroblast growth factor receptor-1-mediated endothelial cell proliferation is dependent on the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk.

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) expressed on endothelial cells leads to cellular migration and proliferation. We have examined the role of the Src homology (SH) 2/SH3 domain-containing adaptor protein Crk in these processes. Transient tyrosine phosphorylation of Crk in fibroblast growth factor-2-stimulated endothelial cells was dependent on the juxtamembrane tyrosine residue 463 in FGFR-1, and a Crk SH2 domain precipitated FGFR-1 via phosphorylated Tyr-463, indicating direct complex formation between Crk and FGFR-1. Furthermore, Crk SH2 and SH3 domains formed ligand-independent complexes with Shc, C3G, and the Crk-associated substrate (Cas). Tyrosine phosphorylation of C3G and Cas increased as a consequence of growth factor treatment. We examined the role of Crk in FGFR-1-mediated cellular responses by use of cells expressing chimeric platelet-derived growth factor receptor-alpha/FGFR-1 (alphaR/FR) wild type and mutant Y463F receptors. The kinase activity of alphaR/FR Y463F was intact, but both Crk and the adaptor FRS-2 were no longer tyrosine-phosphorylated in the mutant cells. Both wild type and mutant receptor cells migrated efficiently, whereas cells expressing the mutant alphaR/FR Y463F failed to proliferate and Erk2 and Jun kinase activities were suppressed in these cells. In wild type alphaR/FR cells transiently expressing an SH2 domain mutant of Crk, Erk and Jun kinase activities as well as DNA synthesis were attenuated. Our data indicate that Crk participates in signaling complexes downstream of FGFR-1, which propagate mitogenic signals.     

A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport

Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein). We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgamma1, the p85 subunit of the phosphoinositide 3-kinase and Crk-II. It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins. We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake. Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process. Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport. In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains. These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake.     

用基因诱捕法制作Ayu17-449基因敲除小鼠模型

为了探明Ayu17-449基因在小鼠生长发育过程中的功能, 用特殊的诱捕载体（Gene trapping vector）导入小鼠ES细胞中,5′RACE、Southern blot方法鉴定成功地单一捕获Ayu17-449基因后,由这种ES制作了Ayu17-449 敲除小鼠并用Northern blot方法该基因在突变小鼠体内的表达。结果在Ayu17-449 敲除小鼠体内,诱捕载体位于Ayu17-449基因的翻译起始密码上游,Ayu17-449基因的转录被抑制。表明Ayu17-449敲除小鼠为分析Ayu17-449基因的功能提供了可靠的实验材料。      

Identification of CrkL-SH3 binding proteins from embryonic murine brain: implications for Reelin signaling during brain development

The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling, which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation, which recruits Crk/CrkL and SH3-bound effectors. Previously, 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multifaceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

Characterization of the CIN85 adaptor protein and identification of components involved in CIN85 complexes

CIN85 is an 85-kDa adaptor protein whose functions in signaling pathways are presently unknown. Using the yeast two-hybrid screen, the B cell linker protein (BLNK) was identified as a binding partner of CIN85. Coimmunoprecipitation experiments using mammalian cells revealed that CIN85 directly bound to BLNK through its SH3 domains. Immunostaining analysis showed that CIN85 and BLNK were colocalized in the cytoplasm. These results indicate a potential role of CIN85 in the B cell receptor-mediated signaling pathway. It was also found that Crk-I, Crk-II, p130(Cas), p85-PI3K, Grb2, and Sos1 were components of CIN85 complexes. CIN85 interacted with itself through its coiled-coil region, resulting in formation of a tetramer. Both the coiled-coil region and SH3 domains of CIN85 were responsible for its subcellular localization. Our data suggest that CIN85 may serve for regulation of various signaling events through formation of its diverse complexes.     

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas–Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.     

Avian and 1918 Spanish influenza a virus NS1 proteins bind to Crk/CrkL Src homology 3 domains to activate host cell signaling

NS1 (nonstructural protein 1) is an important virulence factor of the influenza A virus. We observed that NS1 proteins of the 1918 pandemic virus (A/Brevig Mission/1/18) and many avian influenza A viruses contain a consensus Src homology 3 (SH3) domain-binding motif. Screening of a comprehensive human SH3 phage library revealed the N-terminal SH3 of Crk and CrkL as the preferred binding partners. Studies with recombinant proteins confirmed avid binding of NS1 proteins of the 1918 virus and a representative avian H7N3 strain to Crk/CrkL SH3 but not to other SH3 domains tested, including p85alpha and p85beta. Endogenous CrkL readily co-precipitated NS1 from cells infected with the H7N3 virus. In transfected cells association with CrkL was observed for NS1 of the 1918 and H7N3 viruses but not A/Udorn/72 or A/WSN/33 NS1 lacking this sequence motif. SH3 binding was dispensable for suppression of interferon-induced gene expression by NS1 but was associated with enhanced phosphatidylinositol 3-kinase signaling, as evidenced by increased Akt phosphorylation. Thus, the Spanish Flu virus resembles avian influenza A viruses in its ability to recruit Crk/CrkL to modulate host cell signaling.     

接头蛋白Crk与肿瘤

Crk是一种重要的细胞内信号接头蛋白,含有SH2和SH3结构域,在信号传递过程中不仅承上启下,对信号强度还有着调控作用.迄今主要发现了4种Crk分子,即v Crk、CrkⅠ、CrkⅡ和CrkL(Crk Like).CrkⅡ和CrkL在分子结构上具有高度同源性,但对信号分子却具有不同的偏好性,从而体现出功能差异.本文就接头蛋白Crk的结构、Crk与相互作用蛋白质的作用机制以及与肿瘤的发生发展关系进行了阐述.