Structure of melanoma inhibitory activity protein, a member of a recently identified family of secreted proteins

Melanoma inhibitory activity (MIA) is a 12-kDa protein that is secreted from both chondrocytes and malignant melanoma cells. MIA has been reported to have effects on cell growth and adhesion, and it may play a role in melanoma metastasis and cartilage development. We report the 1.4-Å crystal structure of human MIA, which consists of an Src homology 3 (SH3)-like domain with N- and C-terminal extensions of about 20 aa each. The N- and C-terminal extensions add additional structural elements to the SH3 domain, forming a previously undescribed fold. MIA is a representative of a recently identified family of proteins and is the first structure of a secreted protein with an SH3 subdomain. The structure also suggests a likely protein interaction site and suggests that, unlike conventional SH3 domains, MIA does not recognize polyproline helices.

The dynamins: Redundant or distinct functions for an expanding family of related GTPases?

In the 7 years since dynamin was first isolated from bovine brain in search of novel microtubule-based motors, our understanding of this enzyme has expanded significantly. We now know that brain dynamin belongs to a family of large GTPases, which mediate vesicle trafficking. Furthermore, this enzymatic activity is markedly increased through association with microtubules, acidic phospholipids, and certain regulatory proteins that contain Src homology 3 (SH3) domains. From functional, genetic, and cellular manipulations, it is now generally accepted that dynamin participates in the endocytic uptake of receptors, associated ligands, and plasma membrane following an exocytic event. These observations have confirmed at least one function of dynamin that was predicted from seminal studies on a pleiotropic mutant, shibirets (shits) in Drosophila melanogaster. Of equal interest is the finding that there are multiple dynamin gene products, including two that are expressed in a tissue-specific manner, and they share marked homology with a larger family of distinct but related proteins. Therefore, it is attractive to speculate that the different dynamins may participate in related cellular functions, such as distinct endocytic processes and even secretion. In turn, dynamin could play an important role in cell growth, cell spreading, and neurite outgrowth. The purpose of this review is to enumerate on the expansive dynamin literature and to discuss the nomenclature, expression, and putative functions of this growing and interesting family of proteins.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases.

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.

Sequence and expression of the murine Hoxd-3 homeobox gene.

Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.

Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain.

A novel human cDNA encoding a cytosolic 62-kDa protein (p62) that binds to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner has been cloned. The cDNA is composed of 2074 nucleotides with an open reading frame encoding 440 amino acids. Northern analysis suggests that p62 is expressed ubiquitously in all tissues examined. p62 is not homologous to any known protein in the data base. However, it contains a cysteine-rich region resembling a zinc finger motif, a potential G-protein-binding region, a PEST motif, and several potential phosphorylation sites. Using T7-epitope tagged p62 expression in HeLa cells, the expressed protein was shown to bind to the lck SH2 domain. Deletion of the N-terminal 50 amino acids abolished binding, but mutagenesis of the single tyrosine residue in this region had no effect on binding. Thus, the cloned cDNA indeed encodes the p62 protein, which is a phosphotyrosine-independent ligand for the lck SH2 domain. Its binding mechanism is unique with respect to binding modes of other known ligands for SH2 domains.

The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Mutant forms of growth factor-binding protein-2 reverse BCR-ABL-induced transformation.

Growth factor-binding protein 2 (Grb2) is an adaptor protein that links tyrosine kinases to Ras. BCR-ABL is a tyrosine kinase oncoprotein that is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive leukemias. Grb2 forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to the activation of the Ras protooncogene. In this report we demonstrate that Grb2 mutant proteins lacking amino- or carboxyl-terminal src homology SH3 domains suppress BCR-ABL-induced Ras activation and reverse the oncogenic phenotype. The Grb2 SH3-deletion mutant proteins bind to BCR-ABL and do not impair tyrosine kinase activity. Expression of the Grb2 SH3-deletion mutant proteins in BCR-ABL-transformed Rat-1 fibroblasts and in the human Ph1-positive leukemic cell line K562 inhibits their ability to grow as foci in soft agar and form tumors in nude mice. Furthermore, expression of the Grb2 SH3-deletion mutants in K562 cells induced their differentiation. Because Ras plays an important role in signaling by receptor and nonreceptor tyrosine kinases, the use of interfering mutant Grb2 proteins may be applied to block the proliferation of other cancers that depend in part on activated tyrosine kinases for growth.

Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas.

The focal adhesion kinase (FAK) has been implicated in integrin-mediated signaling events and in the mechanism of cell transformation by the v-Src and v-Crk oncoproteins. To gain further insight into FAK signaling pathways, we used a two-hybrid screen to identify proteins that interact with mouse FAK. The screen identified two proteins that interact with FAK via their Src homology 3 (SH3) domains: a v-Crk-associated tyrosine kinase substrate (Cas), p130Cas, and a still uncharacterized protein, FIPSH3-2, which contains an SH3 domain closely related to that of p130Cas. These SH3 domains bind to the same proline-rich region of FAK (APPKPSR) encompassing residues 711-717. The mouse p130Cas amino acid sequence was deduced from cDNA clones, revealing an overall high degree of similarity to the recently reported rat sequence. Coimmunoprecipitation experiments confirmed that p130Cas and FAK are associated in mouse fibroblasts. The stable interaction between p130Cas and FAK emerges as a likely key element in integrin-mediated signal transduction and further represents a direct molecular link between the v-Src and v-Crk oncoproteins. The Src family kinase Fyn, whose Src homology 2 (SH2) domain binds to the major FAK autophosphorylation site (tyrosine 397), was also identified in the two-hybrid screen.

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

The retinoblastoma protein binds to RIZ, a zinc-finger protein that shares an epitope with the adenovirus E1A protein.

The retinoblastoma protein (Rb) is a target of viral oncoproteins. To explore the hypothesis that viral proteins may be structural mimics of cellular proteins, we have searched cDNA libraries for Rb-binding proteins. We report here the cloning of a cDNA for the protein RIZ from rat and human cells. RIZ is a 250-kDa nuclear protein containing eight zinc-finger motifs. It contains an Rb-binding motif that shares an antigenic epitope with the C terminus of E1A. A domain is conserved between RIZ and the PRDI-BF1/Blimp-1 differentiation factor. Other motifs of RIZ include putative GTPase and SH3 (src homology domain 3) domains. RIZ is preferentially expressed in both adult and embryonic rat neuroendocrine tissues. It is also expressed in human retinoblastoma cells and at low levels in all other human cell lines examined. While the function of RIZ is not yet clear, its structure and pattern of expression suggest a role for RIZ in transcriptional regulation during neuronal differentiation and pathogenesis of retinoblastoma.