Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity

Src homology 2 (SH2) domains are modular protein structures that bind phosphotyrosine (pY)-containing polypeptides and regulate cellular functions through protein-protein interactions. Proteomics analysis showed that the SH2 domains of Src family kinases are themselves tyrosine phosphorylated in blood system cancers, including acute myeloid leukemia, chronic lymphocytic leukemia, and multiple myeloma. Using the Src family kinase Lyn SH2 domain as a model, we found that phosphorylation at the conserved SH2 domain residue Y¹⁹⁴ impacts the affinity and specificity of SH2 domain binding to pY-containing peptides and proteins. Analysis of the Lyn SH2 domain crystal structure supports a model wherein phosphorylation of Y¹⁹⁴ on the EF loop modulates the binding pocket that engages amino acid side chains at the pY+2/+3 position. These data indicate another level of regulation wherein SH2-mediated protein-protein interactions are modulated by SH2 kinases and phosphatases.Src homology 2 (SH2) domains are modular protein structures that are important for signal transduction due to their ability to bind phosphotyrosine (pY)-containing polypeptides within defined amino acid sequence motifs (1). SH2 domains are found in various signaling enzymes and adaptor proteins. Given the reversibility of protein tyrosine phosphorylation and the affinity of SH2-pY binding, the interactions of SH2 domains are inherently dynamic and diverse. Indeed, selective, transient binding to pY motifs is a key mechanism through which intracellular signaling networks are dynamically assembled, localized, and regulated. In addition to mediating protein interactions in trans, SH2 domains bind intramolecularly (2). For example, in Src family kinases (SFKs), the SH2 domain binds in cis to the phosphorylated C-terminal tail as a mechanism to constrain and thereby auto-inhibit the intervening tyrosine kinase domain (3, 4). As well, SH2 domains of cytoplasmic tyrosine kinases have been shown to affect the kinase activity of adjacent kinase domains through allosteric interactions (5). The SFKs are therefore highly regulated as a function of their SH2 domains, which exist in dynamic equilibrium between intra- and intermolecular interactions (6). Hence, as discussed by Pawson (7), the transient and diverse interactions of an SH2 domain can regulate signaling enzymes and constitutes a major mechanism of signal transduction in response to extracellular signals.The structure of the SH2 domain has been extensively characterized. At its core is a conserved antiparallel β-sheet sandwiched between two α-helices (8). SH2 domains bind phosphotyrosine-containing peptides in an extended conformation across the central β-sheet, with the pY residue inserted in a deep recognition pocket formed by conserved residues from strands βB, βC, and βD, helix αA, and the phosphate binding loop. Peptide binding specificity is determined by more variable binding surfaces on the SH2 domain, which recognize residues C-terminal to the pY residue. For the SFK SH2 domains, the three residues C-terminal to the pY residue (pY+1,+2,+3) are dominant determinants of specificity (9, 10), with the domain binding most tightly to sequences containing the motif pYEEI (11, 12). The hydrophobic pY+3 residue inserts in a deep hydrophobic specificity pocket defined by residues of the EF and BG loops (8, 13, 14). Indeed, structural analysis of the SH2 domain revealed that the configuration of the EF and BG loops is critical in dictating SH2 domain specificity by shaping the ligand-binding surface and controlling accessibility of the pY+3 binding pocket (15). Mutation of a single residue of the EF loop can drastically impact peptide binding specificity by altering the pY+3 pocket (15–17), indicating the importance of the pY+3 pocket in substrate selectivity for the SFK SH2 domains.In addition to binding pY-containing polypeptides, SH2 domains themselves may be modulated by phosphorylation. For example, phosphorylation of the Src SH2 domain at conserved Y²¹³ resulted in activation of the cognate kinase domain, possibly by impairing SH2 binding to the phosphorylated C-terminal tail (18). Similarly, phosphorylation of Lck at the equivalent SH2 residue (Y¹⁹²) generally reduced binding to pY-peptides and proteins (19). Phosphorylation at S⁶⁹⁰ in the SH2 domain of the p85α subunit of PI 3-kinase decreased its affinity for pY-containing proteins and promoted feedback inhibition of PI 3-kinase and Akt in response to cellular starvation (20). Conversely, tyrosine phosphorylation of the tensin-3 SH2 domain stimulated substrate binding and biological activity (21). Therefore, phosphorylation of SH2 domains appears to be a general mechanism for modulating their binding properties.Here, we report that Y¹⁹⁴ in the SH2 domain of the SFK Lyn, a residue conserved in SFK SH2 domains, is frequently phosphorylated in hematopoietic and other cancers. In vitro protein and peptide interactions with the Lyn SH2 domain were affected by this phosphorylation. Our results suggest that tyrosine phosphorylation of the SFK SH2 domain modulates both its binding affinity and specificity and may constitute another layer of regulation in signaling networks.     

Construction of minimum size cellulase (Cel5Z) from Pectobacterium chrysanthemi PY35 by removal of the C-terminal region

Pectobacterium chrysanthemi PY35 secretes the endoglucanase Cel5Z, an enzyme of the glycoside hydrolase family 5. Cel5Z is a 426 amino acid, signal peptide (SP)-containing protein composed of two domains: a large N-terminal catalytic domain (CD; 291 amino acids) and a small C-terminal cellulose binding domain (CBD; 62 amino acids). These two domains are separated by a 30 amino acid linker region (LR). A truncated cel5Z gene was constructed with the addition of a nonsense mutation that removes the C-terminal region of the protein. A truncated Cel5Z protein, consisting of 280 amino acid residues, functioned as a mature enzyme despite the absence of the SP, 11 amino acid CD, LR, and CBD region. In fact, this truncated Cel5Z protein showed an enzymatic activity 80% higher than that of full-length Cel5Z. However, cellulase activity was undetectable in mature Cel5Z proteins truncated to less than 280 amino acids.     

Cloning and Characterization of Novel CIS Family Genes

We have reported two JAK-signaling modulators, CIS (cytokine-inducible SH2 protein) and JAB (JAK2 binding protein), which are structurally related. Here we cloned three additional CIS family genes (CIS2, CIS3, and CIS4) on the basis of an expression sequence tag (EST) database search. We also found at least two additional candidates of this gene family in the database. These genes were induced by erythropoietin and granulocyte-macrophage colony stimulating factor in certain hematopoietic cell lines. The SH2 domain and a C-terminal 40 amino acid region, designated the CIS homology domain (CH domain), are highly conserved in this family, while the N-terminal regions of these proteins share little similarity. A yeast two-hybrid assay andin vitroandin vivobinding assays revealed that in addition to JAB, CIS3 bound to the JAK2 tyrosine kinase domain (JH1), although the interaction of CIS3 with the JAK2-JH1 domain was much weaker than that of JAB. Transient expression of JAB and CIS3, but not other CISs, strongly inhibited leukemia inhibitory factor (LIF)-induced STAT3-reporter gene activation in 293 cells. Furthermore, constitutive overexpression of JAB and CIS3 in M1 leukemia cells prevented LIF-induced differentiation and growth arrest. Although the physiological function remains to be investigated, CIS family genes could play a role in the negative regulation of cytokine signaling by interacting with specific targets.     

Microencapsulated bile salt hydrolase producing <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> for oral targeted delivery in the gastrointestinal tract

A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa. The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was 1,982.8 U mg⁻¹. The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction temperature of XynS20 was 45°C; at temperatures below 30 and above 55°C, enzyme activity was less than 50% of that at the optimal temperature.     

Solution structure of N-terminal SH3 domain of Vav and the recognition site for Grb2 C-terminal SH3 domain

The three-dimensional structure of the N-terminal SH3 domain (residues 583–660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.     

Stability and folding of the SH3 domain of bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) plays an important role in B cell development. Deletion of C-terminal 14 amino acids of the SH3 domain of BTK results in X-linked agammaglobulinemia (XLA), an inherited disease. We report here on the stability and folding of SH3 domain of BTK. Peptides corresponding to residues 216–273 (58 residues) and 216–259 (44 residues) of BTK SH3 domain were synthesized by solid phase methods; the first peptide constitutes the entire SH3 domain of BTK while the latter peptide lacks 14 amino acid residues of the C-terminal. The 58 amino acid peptide forms mainly a β-barrel type folding unit. Although small and lacking disulfide bonds, this peptide is extremely stable to thermal denaturation. Based on circular dichroism measurements, its melting temperature was found to be high, 82°C at pH 6.0. However, the Gibbs free energy (ΔG) of the intrinsic stability and thermodynamic spontaneity of unfolding were found to be low, 2.6 kcal/mol by Gdn·HCl denaturation experiments, as compared to 12 kcal/mol obtained for larger single domain proteins, indicating poor stability of SH3 domain. Addition of 500 mM of Na₂SO₄ increased the free energy change ΔG to 4.0 kcal/mol, suggesting an ionic strength effect. The truncated peptide fails to fold correctly and adopts random coil conformation in contrast to 58 amino acid β-barrel peptide, which exhibits high thermal stability but normal or low stability at ambient temperature. These results, to our knowledge the first to delineate the importance of C-terminal in structural integrity of SH3 domains, indicate also that improper folding and/or poor stability of mutant SH3 domain in BTK likely causes XLA. Proteins 28:465–471 © 1996 Wiley-Liss, Inc.     

Sequence conservation between porcine and human LRRK2

Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved and therefore likely functional domains. The LRRK2 cDNA contains an open reading frame of 7,578 bp. The predicted LRRK2 protein consists of 2,526 amino acids of 86–93% identity with its mammalian couterparts. The deduced amino acid sequence of encoded porcine LRRK2 protein displays extensive homology with its human counterpart, with greatest similarities in those regions that contain the kinase domain, the Roc domain and the COR motif. Expression of porcine LRRK2 mRNA in various organs and tissues is similar to its human counterpart and not limited to the brain. The obtained data show that the LRRK2 sequence and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson’s disease. The sequence of the porcine LRRK2 cDNA, encoding the LRRK2/dardarin protein, and the genomic sequence of LRRK2 have been submitted to DDBJ/EMBL/GenBank under the Accessions Numbers EU019992, and EU019994, respectively.     

Exploring the systematic effect of N-substituted PxxP motifs on peptoid affinity to ARHGEF5/TIM SH3 domain and its relationship with ARHGEF5/TIM activation

The TIM protein is a short isoform of full-length Rho guanine nucleotide exchange factor 5 (ARHGEF5), which acts as a functional regulator of Rho-dependent signaling pathways by activating the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a proline-rich region ⁴⁷SSPRQP RKAL⁵⁶ (termed as SSP peptide) between the putative helix and the DH domain. Previously, we demonstrate that the auto-inhibitory state of TIM protein can be relieved by targeting its SH3 domain with rationally designed peptide ligands. However, the designed natural peptides have only a moderately increased affinity (~2-fold) as compared to the cognate SH3-SSP interaction and are susceptible to protease degradation. Here, considering that proline is the only endogenous N-substituted amino acid that plays a critical role in SH3-peptide recognition, the two key proline residues Pro49 and Pro52 in the core ⁴⁹PxxP ⁵² motif of SSP peptide are systematically replaced by 19 N-substituted amino acid types to derive a variety of nonnatural peptoid ligands for TIM SH3 domain. Dynamics and energetics analyses reveal that the replacement would impair the active polyproline II (PPII) helical conformation of SSP peptide due to lack of structural constraint introduced by the five-membered ring of native proline side-chains, thus increasing the peptide flexibility that could incur a large entropy penalty upon binding to the domain. However, the impairment is not very significant and the peptide affinity may also be restored and improved if the N-substituted motif of derived peptiod ligands can effectively interact with the PxxP-binding site of TIM SH3 domain. Consequently, a number of potent peptoids are successfully designed by fluorescence spectroscopy confirmation, in which three (ie, SSP[N-Ile49, N-Asn52], SSP[N-Phe49, N-Gln52], and SSP[N-Tyr49, N-Asn52]) exhibit considerably increased affinity (K_d = 0.09, 0.07, and 0.04 μM, respectively) relative to the native SSP peptide (K_d = 0.87 μM). In addition, guanine nucleotide exchange assays also substantiate that the designed SH3-targeted peptiods can effectively enhance TIM-catalyzed RhoA exchange activity (EA), which is observed to present an exponential relationship with the measured SH3-peptoid binding affinity (pK_d).     

人EEN蛋白的结构预测和功能分析

采用基于神经网络的算法预测了我们自行克隆的新的白血病相关蛋白ＥＥＮ（ｅｘｔｒａ ｅｌｅｖｅｎｎｉｎｅｔｅｅｎ, ＥＥＮ）全长分子的二级结构,结果表明：ＥＥＮ 蛋白可能有三个结构域,Ｎ 端由三段α螺旋和短β折叠组成,中间为四段α螺旋组成的四螺旋结构,Ｃ端为ＳＨ３结构域,类似于在受体酪氨酸激酶信号传导途径中起重要作用的ＳＥＭ－５／ＧＲＢ２ Ｃ端ＳＨ３结构域;利用同源蛋白结构模拟的方法,模拟了ＥＥＮ ＳＨ３结构域的三维结构,结果表明：ＥＥＮ ＳＨ３结构域与ＳＥＭ－５／ＧＲＢ２ ＳＨ３结构域具有相近的结构,构成脯氨酸结合区的氨基酸非常保守．上述结果提示：ＥＥＮ 蛋白可能为新的信号蛋白,可能涉及新的信号传导途径或新的信号传导旁路,ＳＨ３结构域是其功能区域．     

Solution Structure of Tensin2 SH2 Domain and Its Phosphotyrosine-Independent Interaction with DLC-1

Background

Src homology 2 (SH2) domain is a conserved module involved in various biological processes. Tensin family member was reported to be involved in tumor suppression by interacting with DLC-1 (deleted-in-liver-cancer-1) via its SH2 domain. We explore here the important questions that what the structure of tensin2 SH2 domain is, and how it binds to DLC-1, which might reveal a novel binding mode.

Principal Findings

Tensin2 SH2 domain adopts a conserved SH2 fold that mainly consists of five β-strands flanked by two α-helices. Most SH2 domains recognize phosphorylated ligands specifically. However, tensin2 SH2 domain was identified to interact with nonphosphorylated ligand (DLC-1) as well as phosphorylated ligand.

Conclusions

We determined the solution structure of tensin2 SH2 domain using NMR spectroscopy, and revealed the interactions between tensin2 SH2 domain and its ligands in a phosphotyrosine-independent manner.     

Src Homology 2 Domain Containing Protein 5 (SH2D5) Binds the Breakpoint Cluster Region Protein,BCR, and Regulates Levels of Rac1-GTP

SH2D5 is a mammalian-specific, uncharacterized adaptor-like protein that contains an N-terminal phosphotyrosine-binding domain and a C-terminal Src homology 2 (SH2) domain. We show that SH2D5 is highly enriched in adult mouse brain, particularly in Purkinjie cells in the cerebellum and the cornu ammonis of the hippocampus. Despite harboring two potential phosphotyrosine (Tyr(P)) recognition domains, SH2D5 binds minimally to Tyr(P) ligands, consistent with the absence of a conserved Tyr(P)-binding arginine residue in the SH2 domain. Immunoprecipitation coupled to mass spectrometry (IP-MS) from cultured cells revealed a prominent association of SH2D5 with breakpoint cluster region protein, a RacGAP that is also highly expressed in brain. This interaction occurred between the phosphotyrosine-binding domain of SH2D5 and an NxxF motif located within the N-terminal region of the breakpoint cluster region. siRNA-mediated depletion of SH2D5 in a neuroblastoma cell line, B35, induced a cell rounding phenotype correlated with low levels of activated Rac1-GTP, suggesting that SH2D5 affects Rac1-GTP levels. Taken together, our data provide the first characterization of the SH2D5 signaling protein.     

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.     

The Human Hepatocyte Nuclear Factor 3/Fork Head Gene FKHL13: Genomic Structure and Pattern of Expression

We describe the isolation and characterization of the cDNA for FKHL13, the human homologue of the mouse hepatocyte nuclear factor 3/fork headhomologue 4 (HFH-4) gene, a member of the HNF-3/fork head(also called winged helix) gene family. Members of this gene family contain a conserved DNA binding region of approx. 110 amino acids and are thought to play an important role in cell-specific differentiation. Previous analysis of the mouse and rat HFH-4 cDNAs revealed a distinct pattern of expression for this gene, suggesting that the gene plays an important role in the differentiation of lung and oviduct/ampulla epithelial cells and testicular spermatids. Analysis of the human FKHL13 gene confirmed this pattern of expression. We also found expression in adult human brain cortex, which we were able to confirm for the mouse. The expression pattern of FKHL13/HFH-4, confined to cilia/flagella-producing cells, leads us to believe that the gene plays an important role in the regulation of axonemal structural proteins. We show that the human gene for FKHL13 lies on chromosome 17 (comparison with the chromosomal location of the mouse gene strongly suggests 17q22–q25) and that the gene, which is approx. 6 kb, contains a single intron disrupting thefork headDNA binding domain. Such a disruption of a functional unit provides strong evidence for the theory of intron insertion during gene evolution. The expression of the gene is probably controlled by the CpG island, which is located in the promoter region of the gene. We also demonstrate that the FKHL13 gene is highly conserved among a wide variety of species, including birds.