Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.     

Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.     

Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands. Our results indicate that Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.     

The identification of two Drosophila K homology domain proteins. Kep1 and SAM are members of the Sam68 family of GSG domain proteins

Sam68 is a member of a growing family of RNA-binding proteins that contains an extended K homology (KH) domain embedded in a larger domain called the GSG (GRP33, Sam68, GLD1) domain. To identify GSG domain family members, we searched data bases for expressed sequence tags encoding related portions of the Sam68 KH domain. Here we report the identification of two novel Drosophila KH domain proteins, which we termed KEP1 (KH encompassing protein) and SAM. SAM bears sequence identity with mammalian Sam68 and may be the Drosophila Sam68 homolog. We demonstrate that SAM, KEP1, and the recently identified Drosophila Who/How are RNA-binding proteins that are able to self-associate into homomultimers. The GSG domain of KEP1 and SAM was necessary to mediate the RNA binding and self-association. To elucidate the cellular roles of these proteins, SAM, KEP1, and Who/How were expressed in mammalian and Drosophila S2 cells. KEP1 and Who/How were nuclear and SAM was cytoplasmic. The expression of KEP1 and SAM, but not Who/How, activated apoptotic pathways in Drosophila S2 cells. The identification of KEP1 and SAM implies that a large GSG domain protein family exists and helps redefine the boundaries of the GSG domain. Taken together, our data suggest that KEP1 and SAM may play a role in the activation or regulation of apoptosis and further implicate the GSG domain in RNA binding and oligomerization.     

The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr

bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.     

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.     

Shc interaction with Src homology 2 domain containing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding domain and two specific phosphotyrosines on SHIP

The adapter protein Shc has been implicated in mitogenic signaling via growth factor receptors, cytokine receptors, and antigen receptors on lymphocytes. Besides the well characterized interaction of Shc with molecules involved in Ras activation, Shc also associates with a 145-kDa tyrosine-phosphorylated protein upon triggering via antigen receptors and many cytokine receptors. This 145-kDa protein has been recently identified as an SH2 domain containing 5'-inositol phosphatase (SHIP) and has been implicated in the regulation of growth and differentiation in hematopoietic cells. In this report, we have addressed the molecular details of the interaction between Shc and SHIP in vivo. During T cell receptor signaling, tyrosine phosphorylation of SHIP and its association with Shc occurred only upon activation. We demonstrate that the phosphotyrosine binding domain of Shc is necessary and sufficient for its association with tyrosine-phosphorylated SHIP. Through site-directed mutagenesis, we have identified two tyrosines on SHIP, Tyr-917, and Tyr-1020, as the principal contact sites for the Shc-phosphotyrosine binding domain. Our data also suggest a role for the tyrosine kinase Lck in phosphorylation of SHIP. We also show that the SH2 domain of SHIP is dispensable for the Shc-SHIP interaction in vivo. These data have implications for the localization of the Shc.SHIP complex and regulation of SHIP function during T cell receptor signaling.     

Structural determinants of the interaction between the erbB2 receptor and the Src homology 2 domain of Grb7

The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the betaD6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the betaD6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.     

Platelet endothelial cell adhesion molecule-1 is phosphorylatable by c-Src, binds Src-Src homology 2 domain, and exhibits immunoreceptor tyrosine-based activation motif-like properties

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and in in vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 --> Phe and Tyr686 --> Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.     

Tyrosine phosphorylation-dependent and -independent associations of protein kinase C-delta with Src family kinases in the RBL-2H3 mast cell line: regulation of Src family kinase activity by protein kinase C-delta

Src kinases and protein kinase C (PKC) have been well studied for their role in oncogenic and normal cellular processes. Herein we report on a novel regulatory pathway mediated by the interaction of PKC-delta with p53/56Lsy (Lyn) and with p60Src (Src) that results in the phosphorylation and increased activity of Lyn and Src. In the RBL-2H3 mast cell line, the interaction of PKC-delta with Lyn required the activation of the high affinity receptor for IgE (FcsigmaRI) while the interaction with Src was constitutive. Increased complex formation of PKC-delta with Lyn or Src led to increased serine phosphorylation and activity of the Src family kinases. Conversely, Lyn was found to phosphorylate Lyn-associated and recombinant PKC-delta in vitro and the tyrosine 52 phosphorylated PKC-delta was recruited to associate with the Lyn SH2 domain. The constitutive association of PKC-delta with Src did not result in the tyrosine phosphorylation of PKC-delta prior to or after FsigmaRI engagement. However in cells over-expressing PKC-delta, FsigmaRI engagement resulted in the dramatic inhibition of Src activity and some inhibition of Lyn activity. Thus, the interaction and cross-talk of PKC-delta with Src family kinases suggests a novel and inter-dependent mechanism for regulation of enzymatic activity that may serve an important role in cellular responses.     

Temporal phases in apoptosis defined by the actions of Src homology 2 domains, ceramide, Bcl-2, interleukin-1beta converting enzyme family proteases, and a dense membrane fraction

We have begun to explore the mechanisms of apoptosis using a cell-free system based on extracts from Xenopus eggs. Nuclei assembled or placed in these extracts undergo the morphological changes typical of apoptosis and eventually disintegrate. We used this system to investigate the potential involvement in apoptosis of proteins containing Src homology 2 (SH2) domains, which are known to interact with specific tyrosine-phosphorylated ligands. SH2 domains from a number of signaling proteins, including Lck, Src, and Abl, inhibited apoptosis when present at concentrations of 10-100 nM. The inhibition was dependent on specific interaction with endogenous tyrosine-phosphorylated ligands. A synthetic peptide ligand for Src family SH2 domains also inhibited apoptosis in a phosphotyrosine-dependent manner. Kinetic analysis defined three phases in the apoptotic process occurring in this cell-free system. SH2 domains and ceramide act throughout the first 60-90 min of the process (the "initiation" phase). Next, Bcl-2, interleukin-1beta converting enzyme family(CPP32-like) proteases, and the heavy membrane fraction act in a period occurring approximately 90-120 min after the start of incubation (the "sentencing" phase). In the final phase ("execution"), the process of active nuclear destruction ensues.     

Small molecule inhibitors of the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and Src family tyrosine kinases

The inhibition of tyrosine kinases involved in growth factor signal transduction pathways represents an attractive strategy for controlling aberrant cellular growth. Over the last 4-5 years, there have been numerous reports on the discovery of small molecule inhibitors for potential therapeutic applications to a number of proliferative diseases, principally cancer and restenosis, where the over-expression of certain tyrosine kinases has been demonstrated. These include, amongst others, the platelet-derived growth factor receptor, the fibroblast growth factor receptor, and the nonreceptor c-Src tyrosine kinase. This review compiles published reports and patent filings from 1995 to mid-1997 that include data directly related to inhibition of the platelet-derived growth factor receptor, fibroblast growth factor receptor, and Src family tyrosine kinases. Potential clinical applications for selected classes of tyrosine kinase inhibitors reviewed herein will likely depend on the demonstration of meaningful activity in a variety of therapeutic targets in animal models.     

Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice

All Src family non-receptor tyrosine kinases are negatively regulated by phosphorylation at a carboxy-terminal tyrosine. To analyze the significance of this regulation during development, we have generated mice deficient in Csk, a kinase that phosphorylates this tyrosine, by gene targeting in embryonic stem cells. Homozygous mutant embryos exhibit a complex phenotype that includes defects in the neural tube and die between day 9 and day 10 of gestation. Cells derived from these embryos exhibit an order of magnitude increase in activity of Src and the related Fyn kinase. Phosphorylation at the carboxy-terminal tyrosine of Src was reduced but not eliminated and was accompanied by increased phosphorylation at another key tyrosine residue. These results demonstrate that Src family kinase activity is critically dependent on phosphorylation by Csk and suggest that the regulation of kinase activity may be essential during embryogenesis.     

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.     

Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1)

A fragment consisting of residues 584-1071 of the mouse Son-of-sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1. Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells. Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments. First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions. On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain. By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions. This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor. The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain. Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.     

Identification and sequence analysis of two new members of the SKALP/elafin and SPAI-2 gene family. Biochemical properties of the transglutaminase substrate motif and suggestions for a new nomenclature

The human epithelial proteinase inhibitor SKALP/elafin and the porcine sodium-potassium ATPase inhibitor SPAI-2 are two highly homologous proteins that share an NH2-terminal transglutaminase substrate domain and a COOH-terminal whey acidic protein (WAP) domain. Here we describe the bovine and simian orthologs of SKALP/elafin as well as two new bovine family members that are designated Trappin-4 and Trappin-5 on the basis of a new nomenclature that we propose (Trappin = TRansglutaminase substrate and WAP motif-containing ProteIN). Sequence analysis of Trappin-4 and Trappin-5 revealed a domain structure that is very similar to SPAI-2 (Trappin-1) and SKALP/elafin (Trappin-2). The transglutaminase substrate motifs are conserved although the number of repeats varies among species and among family members. The sequence of Trappin-4 and Trappin-5 diverges from Trappin-1 and Trappin-2 at the putative reactive site in the WAP domain. The bovine ortholog of Trappin-2 is expressed in tongue and snout epidermis; Trappin-4 is expressed in trachea, ileum, and tongue; and Trappin-5 is expressed at low levels in trachea, as determined by RNase protection and Northern blot analysis. Based on the analysis of 67 transglutaminase substrate repeats as present in all known Trappin gene family members from four different mammalian species a consensus sequence could be established: Gly-Gln-Asp-Pro-Val-Lys (GQDPVK). Using biotinylated hexapeptide probes we found that the GQDPVK sequence is a very efficient transglutaminase substrate both for guinea pig liver transglutaminase and for epidermal transglutaminase, and it acts as acyl donor as well as acceptor. We propose that the Trappin protein family forms a new group of enzyme inhibitors with various specificities of the WAP domain, which share transglutaminase substrate motifs that can act as an anchoring sequence.