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Biomedical subjects

H Hanafusa

Publications and source records attributed to H Hanafusa.

At least 73 records · Page 4Linked to original sources
PubMed1995

The SH3 domain of Crk binds specifically to a conserved proline-rich motif in Eps15 and Eps15R.

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Adaptor Proteins, Signal Transducing↗
PubMed1995

Cellular proteins binding to the first Src homology 3 (SH3) domain of the proto-oncogene product c-Crk indicate Crk-specific signaling pathways.

The widely expressed c-Crk protein, composed of one SH2 and two SH3 domains, lacks an apparent catalytic domain, suggesting that it functions through the formation of specific complexes with other proteins. Bacterially expressed c-Crk formed in vitro highly stable complexes via the first SH3 domain [SH3(N)]. Most prominent were a 185 kDa protein of unknown identity (p185), Sos- immunoreactive bands of 170 kDa (p170) and 145 to 155 kDa bands, corresponding to the recently cloned C3G protein. p170 also bound to Ash/Grb2 and Nck while p185 and C3G bound only to Crk. Additional Crk binding proteins were found in hematopoietic cells, particularly the myeloid-monocytic lineage. The protein binding properties of Crk were subsequently compared to CRKL, the product of a homologous but distinct gene, and found to be very similar. The binding of two guanine nucleotide exchange factors, Sos and C3G, to Crk and CRKL indicates that Ras or related proteins likely play a role in signaling through Crk family proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1995

A novel ligand for SH3 domains. The Nck adaptor protein binds to a serine/threonine kinase via an SH3 domain.

We have previously shown that overexpression of the SH2- and SH3-containing Nck adaptor protein causes transformation of mammalian fibroblast. To elucidate the mechanism by which it deregulates growth, we have sought to identify potential effectors for Nck. We report that a serine/threonine kinase, which we term NAK (for Nck-associated kinase), associates with Nck in vivo and in vitro. Using glutathione S-transferase fusion proteins generated with isolated domains of Nck, we demonstrate that NAK binds specifically to the second of Nck's three SH3 domains. NAK is complexed with Nck in a wide variety of cell types, including NIH3T3, A431, PC12, and Hela cells.

3T3 Cells↗
PubMed1995

Activation of YRP kinase by v-Src and protein kinase C-mediated signal transduction pathways.

We have previously reported that a serine(threonine) protein kinase that phosphorylates histone H1 in vitro is activated by tyrosine phosphorylation in v-Src-transformed rat 3Y1 fibroblasts. We now refer to this kinase as YRP kinase, for tyrosine-regulated protein kinase. Since YRP kinase may play a role in mediating the growth-stimulatory and morphology-altering effects of v-Src, we have further examined the signal transduction involved in the activation of YRP kinase. Although YRP kinase is constitutively activated in fibroblasts transformed by v-Src, activation of protein kinase C was also found to lead to activation of YRP kinase. Activation of YRP kinase by protein kinase C was found to be potentiated by vanadate treatment or overexpression of c-Src. The activation of YRP kinase by v-Src, however, does not appear to be mediated by protein kinase C, suggesting that YRP kinase can be activated by two separate signal transduction pathways. Transformation of fibroblasts by v-Ras or v-Mil did not result in activation of YRP kinase, indicating that the MAP kinase pathway does not mediate the activation of YRP kinase by v-Src or protein kinase C.

Animals↗
PubMed1995

Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of src.

To search for the biochemical properties of cells relevant to transformation by p60v-src, we examined the activities and amounts of metalloproteinase (gelatinase) released from chicken embryonic fibroblasts infected with various mutants of Rous sarcoma virus by zymography and immunoblotting. While nontransforming Src proteins, including cellular p60c-src and its nonmyristylated form, had no stimulatory effect, wild-type p60v-src and the transforming mutant of cellular p60c-src stimulated the secretion and proteolytic activation of metalloproteinases. Moreover, the activation of metalloproteinase secretion strongly correlated with the invasiveness of cells assayed by the modified Boyden Chamber method. Chimeric mutants between v-src and c-src, which are transforming but produce less distinct morphological changes in infected cells, also stimulated the secretion of metalloproteinases as well as wild-type p60v-src. Deletion mutants of v-Src in which varying portions of the NH2-terminal half of p60v-src are deleted stimulated secretion to a level similar to that of wild-type regardless of the degree of morphological change they induce. Together with Src protein, other oncogene products including Yes, Fps, ErbB and Crk were also found to stimulate the secretion of metalloproteinases. Thus, these results suggest that transformation of cells with src and other oncogenes is closely associated with the enhanced secretion of metalloproteinases that may play an important role in tumor invasion and metastasis.

Animals↗
PubMed1995

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

BACKGROUND: Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS: In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Amino Acid Sequence↗
PubMed1994

Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk.

The widely expressed cellular Crk protein has the domain structure SH2-SH3-SH3. We have previously demonstrated that the more N-terminal SH3 domain of Crk (CrkSH3(N)) specifically binds several cytoplasmic proteins. A cDNA encoding one of these proteins was isolated and found to have two different splice forms. The sequence is virtually identical to C3G, a guanine-nucleotide exchange factor. The center region of the 145-155-kDa protein contains four similar proline-rich sequences which are capable of binding individually to the SH3(N) domains of c-Crk and v-Crk. Comparison of these sequences in C3G to proline-rich sequences in other Crk-binding proteins suggests that positively charged amino acids following the prolines play an important role in the binding to the CrkSH3(N) domain. The endogenous C3G could be coprecipitated with Crk from cell lysates of cells expressing high levels of c-Crk or v-Crk, suggesting high binding affinity and a possible interaction in vivo. Unlike many other SH3-binding proteins which interact with multiple SH3 domains, C3G from cell lysates binds preferentially to the CrkSH3(N) domain. This unique binding specificity supports the idea that C3G plays an important role in Crk signaling pathways.

Alternative Splicing↗
PubMed1994

Comparative study of three protein-tyrosine phosphatases. Chicken protein-tyrosine phosphatase lambda dephosphorylates c-Src tyrosine 527.

To examine the substrate preference of protein tyrosine phosphatases (PTPs), we compared the activity of three transmembrane PTPs on dephosphorylation and regulation of c-Src and v-Src: chicken PTP lambda (ChPTP lambda), chicken PTP alpha (ChPTP alpha), and human leukocyte common antigen-related molecule (HLAR). In vitro, all three PTPs dephosphorylated v-Src, but only ChPTP lambda dephosphorylated c-Src. Their activities were also compared in Cos cells coexpressing Src and the phosphatase domains of three PTPs. These domains were fused with peptides for myristylation, so they associated with the cellular membrane. When c-Src was coexpressed with myrPTP lambda, its kinase activity was elevated 3-4-folds. This activation was less obvious when c-Src was coexpressed with myrPTP alpha or myrLAR. Analysis by cyanogen bromide cleavage showed that ChPTP lambda and myrPTP lambda dephosphorylated Tyr-527 of c-Src. Our data demonstrated the different activities of three PTPs on phosphoproteins, suggesting that Src Tyr-527 may require more specific PTP(s) than Src Tyr-416 for dephosphorylation in vivo.

Amino Acid Sequence↗
PubMed1994

c-Abl kinase regulates the protein binding activity of c-Crk.

c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation.

3T3 Cells↗
PubMed1994

A transmembrane protein-tyrosine phosphatase contains spectrin-like repeats in its extracellular domain.

We report the first chicken transmembrane protein-tyrosine phosphatase, ChPTP lambda, isolated from a brain cDNA library and preB cells. ChPTP lambda has transcripts of about 5.6 kilobases and is abundant in spleen, intestine, and fibroblasts transformed by oncogenic ras or erbA/B. It has five alternative splicing products varying near their amino termini, and the largest one contains 1237 amino acids. The extracellular domain of ChPTP lambda has several features including a Ser/Thr/Pro-rich region, one fibronectin type III module, and spectrin-like repeats (the first case that spectrin-like repeats were found in the non-cytoplasmic compartment). These repeats were also found in other phosphatases, including CD45 and yeast acid phosphatases PHO5 and PHO3. Antibodies to ChPTP lambda recognized several protein species whose M(r) range from 170,000 to 210,000. ChPTP lambda exhibited phosphotyrosine-specific phosphatase activity. Since CD45 also has these features in the extracellular domain and the two protein-tyrosine phosphatases share 70% similarity in the intracellular domains, we propose that ChPTP lambda and CD45 belong to the same gene family.

Alternative Splicing↗
PubMed1994

Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix.

Animals↗
PubMed1994

The proto-oncogene of v-eyk (v-ryk) is a novel receptor-type protein tyrosine kinase with extracellular Ig/GN-III domains.

v-ryk is the oncogene in the avian acute oncogenic retrovirus RPL30, its cellular counterpart, c-ryk, is described in this report. To avoid the confusion caused by another gene with the same name, we renamed v-ryk to be v-eyk, thereby changing the proto-oncogene's name to c-eyk. c-eyk is expressed highly in chicken spleen and moderately in many other tissues including embryonic tissues. Its cDNA is 3061 base pairs encoding a receptor-type protein tyrosine kinase (PTK) of 974 amino acids. Compared to c-Eyk, v-Eyk is a truncated PTK lacking the extracellular and transmembrane regions of c-Eyk in addition to two amino acid changes. c-Eyk has two C2-type Ig domains and two FN-III domains in its extracellular region, forming a new subfamily of receptor-type PTK together with UFO/Axl/Ark. As suggested by Ig/Fn-III domains in many cell adhesion molecules, this subfamily of PTK may mediate cell-cell interaction.

Amino Acid Sequence↗
PubMed1994

SH2 and SH3 domains as molecular adhesives: the interactions of Crk and Abl.

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

Adhesiveness↗
PubMed1994

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures.

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage-independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage-independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.

Actins↗
PubMed1994

Functional analysis of Csk in signal transduction through the B-cell antigen receptor.

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.

Animals↗