Adhesion structures in leukemia cells and their regulation by Src family kinases

Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.     

The Src,Syk, and Tec family kinases: Distinct types of molecular switches

The Src, Syk, and Tec family kinases are three of the most well characterized tyrosine kinase families found in the human genome. Members of these kinase families function downstream of antigen and F_c receptors in hematopoietic cells and transduce signals leading to calcium mobilization, altered gene expression, cytokine production, and cell proliferation. Over the last several years, structural and biochemical studies have begun to uncover the molecular mechanisms regulating activation of these kinases. It appears that each kinase family functions as a distinct type of molecular switch. This review discusses the activation of the Src, Syk, and Tec kinases from the perspective of structure, phosphorylation, allosteric regulation, and kinetics. The multiple factors that regulate the Src, Syk, and Tec families illustrate the important role played by each of these kinases in immune cell signaling.     

An electrostatic network and long-range regulation of Src kinases

The regulatory mechanism of Src tyrosine kinases includes conformational activation by a change in the catalytic domain tertiary structure and in domain-domain contacts between the catalytic domain and the SH2/SH3 regulatory domains. The kinase is activated when tyrosine phosphorylation occurs on the activation loop, but without phosphorylation of the C-terminal tail. Activation also occurs by allostery when contacts between the catalytic domain (CD) and the regulatory SH3 and SH2 domains are released as a result of exogenous protein binding. The aim of this work is to examine the proposed role of an electrostatic network in the conformational transition and to elucidate the molecular mechanism for long-range, allosteric conformational activation by using a combination of experimental enzyme kinetics and nonequilibrium molecular dynamics simulations. Salt dependence of the induction phase is observed in kinetic assays and supports the role of an electrostatic network in the transition. In addition, simulations provide evidence that allosteric activation involves a concerted motion coupling highly conserved residues, and spanning several nanometers from the catalytic site to the regulatory domain interface to communicate between the CD and the regulatory domains.     

Activation of Src family kinases by colony stimulating factor-1, and their association with its receptor.

The receptor for the macrophage colony stimulating factor-1 (CSF-1R) is a transmembrane glycoprotein with intrinsic tyrosine kinase activity. CSF-1 stimulation promotes the growth of cells of the macrophage lineage and of fibroblasts engineered to express CSF-1R. We show that CSF-1 stimulation resulted in activation of three Src family kinases, Src, Fyn and Yes. Concomitant with their activation, all three Src family kinases were found to associate with the ligand-activated CSF-1 receptor. These interactions were also demonstrated in SF9 insect cells co-infected with viruses encoding the CSF-1 receptor and Fyn, and the isolated SH2 domain of Fyn was capable of binding the CSF-1R in vitro. Analysis of mutant CSF-1Rs revealed that the 'kinase insert' (KI) domain of CSF-1R was not required for interactions with Src family kinases, but that mutation of one of the receptor autophosphorylation sites, Tyr809, reduced both their binding and enzymatic activation. Because fibroblasts expressing this receptor mutant are unable to form colonies in semi-solid medium or to grow in chemically defined medium in the presence of CSF-1, the Src family kinases may play a physiological role in the mitogenic response to CSF-1.     

Regulation of Btk by Src family tyrosine kinases.

Loss of function of Bruton's tyrosine kinase (Btk) results in X-linked immunodeficiencies characterized by a broad spectrum of signaling defects, including those dependent on Src family kinase-linked cell surface receptors. A gain-of-function mutant, Btk*, induces the growth of fibroblasts in soft agar and relieves the interleukin-5 dependence of a pre-B-cell line. To genetically define Btk signaling pathways, we used a strategy to either activate or inactivate Src family kinases in fibroblasts that express Btk*. The transformation potential of Btk* was dramatically increased by coexpression with a partly activated c-Src mutant (E-378 --> G). This synergy was further potentiated by deletion of the Btk Src homology 3 domain. Downregulation of Src family kinases by the C-terminal Src kinase (Csk) suppressed Btk* activation and biological potency. In contrast, kinase-inactive Csk (K-222 --> R), which functioned as a dominant negative molecule, synergized with Btk* in biological transformation. Activation of Btk* correlated with increased phosphotyrosine on transphosphorylation and autophosphorylation sites. These findings suggest that the Src and Btk kinase families form specific signaling units in tissues in which both are expressed.     

The role of Src family kinases in egg activation

Solitary amoebae of Dictyostelium discoideum are frequently exposed to stressful conditions in nature, and their multicellular development is one response to environmental stress. Here we analyzed an aggregation stage abundant gene, krsA, homologous to human krs1 (kinase responsive to stress 1) to understand the mechanisms for the initiation of development and cell fate determination. The krsA- cells exhibited reduced viability under hyperosmotic conditions. They produced smaller aggregates on membrane filters and did not form aggregation streams on a plastic surface under submerged starvation conditions, but were normal in sexual development. During early asexual development, the expression of cAMP-related genes peaked earlier in the knockout mutants. Neither cAMP oscillation in starved cells nor an increase in the cAMP level following osmotic stress was observed in krsA-. The nuclear export signal, as well as the kinase domain, in KrsA was necessary for stream formation. These results strongly suggest that krsA is involved in cAMP relay, and that signaling pathways for multicellular development have evolved in unison with the stress response.     

Activation of Src family tyrosine kinases by ferric ions

The Src-family tyrosine kinases (SFKs) are oncogenic enzymes that contribute to the initiation and progression of many types of cancer. In normal cells, SFKs are kept in an inactive state mainly by phosphorylation of a consensus regulatory tyrosine near the C-terminus (Tyr⁵³⁰ in the SFK c-Src). As recent data indicate that tyrosine modification enhances binding of metal ions, the hypothesis that SFKs might be regulated by metal ions was investigated. The c-Src C-terminal peptide bound two Fe^3 + ions with affinities at pH 4.0 of 33 and 252 μM, and phosphorylation increased the affinities at least 10-fold to 1.4 and 23 μM, as measured by absorbance spectroscopy. The corresponding phosphorylated peptide from the SFK Lyn bound two Fe^3 + ions with much higher affinities (1.2 pM and 160 nM) than the Src C-terminal peptide. Furthermore, when Lyn or Hck kinases, which had been stabilised in the inactive state by phosphorylation of the C-terminal regulatory tyrosine, were incubated with Fe^3 + ions, a significant enhancement of kinase activity was observed. In contrast Lyn or Hck kinases in the unphosphorylated active state were significantly inhibited by Fe^3 + ions. These results suggest that Fe^3 + ions can regulate SFK activity by binding to the phosphorylated C-terminal regulatory tyrosine.     

Involvement of the SH3 domain in Ca2+-mediated regulation of Src family kinases

When cells are treated with Ca(2+) and Ca(2+)-ionophore, c-Src kinase activity increases, whereas c-Yes kinase activity decreases. This opposite modulation can be reproduced in an in vitro reconstitution assay and is dependent on Ca(2+) and on soluble factors present in cell lysates. Since c-Src and c-Yes share a high degree of homology, with the exception of their N-terminal "unique" domains, their activity was thought to be coordinately regulated. To assess the mechanism of regulation we generated stable cell lines expressing eight different constructs containing wild type c-Src and c-Yes, as well as swaps of the unique domain alone, unique and Src homology 3 (SH3) domains together and the SH3 domain alone. Swapping of the unique domains was not sufficient to reverse the regulation of the chimeric molecules. On the other hand, chimeras containing swaps of the unique plus the SH3 domains displayed reverse regulation, implicating both domains in the regulation of kinase activity by Ca(2+). To rule out the participation of the unique domain, we used chimeric molecules with swapped SH3 domains only and found that the SH3 domain is necessary and sufficient to confer Ca(2+)-mediated regulation of Src and Yes tyrosine kinases.     

Structure-function relationships in Src family and related protein tyrosine kinases

There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains.     

EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase

Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as "receptor-like" signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Here we show that Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. Our data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling.     

Kinase pathways in chemoattractant-induced degranulation of neutrophils: the role of p38 mitogen-activated protein kinase activated by Src family kinases

The aim of the present study was to investigate the role of tyrosine phosphorylation pathways in fMLP-induced exocytosis of the different secretory compartments (primary and secondary granules, as well as secretory vesicles) of neutrophils. Genistein, a broad specificity tyrosine kinase inhibitor, blocked the exocytosis of primary and secondary granules, but had only a marginal effect on the release of secretory vesicles. Genistein also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPK), raising the possibility that inhibition of ERK and/or p38 MAPK might be responsible for the effect of the drug on the degranulation response. Indeed, SB203580, an inhibitor of p38 MAPK, decreased the release of primary and secondary granules, but not that of secretory vesicles. However, blocking the ERK pathway with PD98059 had no effect on any of the exocytic responses tested. PP1, an inhibitor of Src family kinases, also attenuated the release of primary and secondary granules, and neutrophils from mice deficient in the Src family kinases Hck, Fgr, and Lyn were also defective in secondary granule release. Furthermore, activation of p38 MAPK was blocked by both PP1 and the hck-/-fgr-/-lyn-/- mutation. Taken together, our data indicate that fMLP-induced degranulation of primary and secondary granules of neutrophils is mediated by p38 MAPK activated via Src family tyrosine kinases. Although piceatannol, a reportedly selective inhibitor of Syk, also prevented degranulation and activation of p38 MAPK, no fMLP-induced phosphorylation of Syk could be observed, raising doubts about the specificity of the inhibitor.     

Src family kinases directly regulate JIP1 module dynamics and activation

JIP1 is a mammalian scaffold protein that assembles and participates in regulating the dynamics and activation of components of the mixed-lineage kinase-dependent JNK module. Mechanisms governing JIP1-JNK module regulation remain unclear. JIP1 is a multiply phosphorylated protein; for this reason, it was hypothesized that signaling by unidentified protein kinases or phosphatases might determine module function. We find that Src family kinases directly bind and tyrosine phosphorylate JIP1 under basal conditions in several naturally occurring systems and, by doing so, appear to provide a regulated signal that increases the affinity of JIP1 for DLK and maintains the JIP-JNK module in a catalytically inactive state.     

Novel regulation of Na, K-ATPase by Src tyrosine kinases in cortical neurons

The Na+, K+-ATPase or Na+, K+-pump plays a critical role in ion homeostasis and many cellular events. The Na+, K+-pump activity is regulated by serine/threonine phosphorylation, the role of tyrosine kinases in the regulation, however, is obscure. We now present novel evidence showing that tyrosine phosphorylation activates the Na+, K+-pump in cortical neurons. The electrogenic activity of the Na+, K+-pump was measured using whole-cell voltage clamp. A tonic activity was revealed by an inward current induced by the specific inhibitor ouabain or strophanthidin; an outward current due to activation of the pump was triggered by raising extracellular K+. The inward and outward currents were attenuated by the tyrosine kinase inhibitor genistein, herbimycin A, or lavendustin A, while blocking tyrosine phosphatases increased the pump current. Down-regulation of the pump current was also seen with the Src inhibitor PP1 and intracellularly applied anti-Lyn or anti-Yes antibody. Consistently, intracellular application of Lyn kinase up-regulated the pump current. Immunoprecipitation and western blotting showed tyrosine phosphorylation and a direct interaction between Lyn and the alpha3 subunit of the Na+, K+-pump. The tyrosine phosphorylation of the alpha3 subunit was reduced by serum deprivation. These data suggest that the Na+, K+-ATPase activity in central neurons is regulated by specific Src tyrosine kinases via a protein-protein mechanism and may play a role in apoptosis.     

Src family kinases as mediators of endothelial permeability: effects on inflammation and metastasis

Src family kinases (SFKs) are signaling enzymes that have long been recognized to regulate critical cellular processes such as proliferation, survival, migration, and metastasis. Recently, considerable work has elucidated mechanisms by which SFKs regulate normal and pathologic processes in vascular biology, including endothelial cell proliferation and permeability. Further, when inappropriately activated, SFKs promote pathologic inflammatory processes and tumor metastasis, in part through their effects on the regulation of endothelial monolayer permeability. In this review, we discuss the roles of aberrantly activated SFKs in mediating endothelial permeability in the context of inflammatory states and tumor cell metastasis. We further summarize recent efforts to translate Src-specific inhibitors into therapy for systemic inflammatory conditions and numerous solid organ cancers. The authors’ own research was supported in part by NIH U54 CA 090810 and P20 CA101936 (G.E.G) and NIH T32 CA 09599 (M.P.K.)