Anti-Lyn Anticorpo [LYN-01] (A86683)

c-Src kinase is a rate-limiting activator of osteoclast (OC) function and Src inhibitors are therefore candidate antiosteoporosis drugs. By affecting alphavbeta3 and macrophage-colony stimulating factor (M-CSF)-induced signaling, c-Src is central to osteoclast activity, but not differentiation. We find Lyn, another member of Src family kinases (SFK) is, in contrast, a negative regulator of osteoclastic bone resorption. The absence of Lyn enhances receptor activator of NF-kappaB ligand (RANKL)-mediated differentiation of osteoclast precursors without affecting proliferation and survival, while its overexpression decreases osteoclast formation. In further contrast to c-Src, Lyn deficiency does not impact the activity of the mature cell. Reflecting increased osteoclast development in vitro, Lyn-/- mice undergo accelerated osteoclastogenesis and bone loss, in vivo, in response to RANKL. Mechanistically, Lyn forms a complex with receptor activator of NF-kappaB (RANK), the tyrosine phosphatase, SHP-1, and the adapter protein, Grb2-associated binder 2 (Gab2). Upon RANKL exposure, Gab2 phosphorylation, JNK, and NF-kappaB activation are enhanced in Lyn-/- osteoclasts, all critical events in osteoclast development. We therefore establish that Lyn regulates osteoclast formation and does it in a manner antithetical to that of c-Src. The most pragmatic aspect of our findings is that successful therapeutic inhibition of c-Src, in the context of the osteoclast, will require its stringent targeting.

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

Signaling through glutamate receptors has been reported in human cancers, but the molecular mechanisms are not fully delineated. We report that in hepatocellular carcinoma and clear cell renal carcinoma cells, increased activity of hypoxia-inducible factors (HIFs) due to hypoxia or VHL loss-of-function, respectively, augmented release of glutamate, which was mediated by HIF-dependent expression of the SLC1A1 and SLC1A3 genes encoding glutamate transporters. In addition, HIFs coordinately regulated expression of the GRIA2 and GRIA3 genes, which encode glutamate receptors. Binding of glutamate to its receptors activated SRC family kinases and downstream pathways, which stimulated cancer cell proliferation, apoptosis resistance, migration and invasion in different cancer cell lines. Thus, coordinate regulation of glutamate transporters and receptors by HIFs was sufficient to activate key signal transduction pathways that promote cancer progression.

Integrins are type I membrane and heterodimeric (alphabeta) cell adhesion receptors. Intracellular signals triggered by ligand-bound integrins are important for cell growth, differentiation, and migration. Integrin alpha(M)beta(2) plays key roles in myeloid cell adhesion, phagocytosis, and degranulation. In this study, we show that protein kinase C (PKC) delta is involved in alpha(M)beta(2) signaling. In human monocytic U937 cells and peripheral blood monocytes, alpha(M)beta(2) clustering induced PKCdelta translocation to the plasma membrane, followed by Tyr(311) phosphorylation and activation of PKCdelta by the src family kinases Hck and Lyn. Interestingly, alpha(M)beta(2)-induced PKCdelta Tyr(311) phosphorylation was not mediated by the tyrosine kinase Syk, which is a well reported kinase in beta(2) integrin signaling. Analysis of the beta(2) cytoplasmic tail showed that the sequence Asn(727)-Ser(734) is important in alpha(M)beta(2)-induced PKCdelta Tyr(311) phosphorylation. It has been shown that alpha(M)beta(2) clustering regulates the expression the transcription factor Foxp1 that has a role in monocyte differentiation. We show that Foxp1 expression was reduced in monocytes that were allowed to adhere to human microvascular endothelial cells. However, the expression of Foxp1 was not affected in monocytes that were treated with PKCdelta-targeting small interfering RNA, suggesting that PKCdelta regulates Foxp1 expression. These results demonstrate a role of PKCdelta in alpha(M)beta(2)-mediated Foxp1 regulation in monocytes.

Lyn kinase, a member of the Src family of tyrosine kinases, functions as both a positive and negative regulator of B cell activation. In the absence of Lyn, BCR signaling is unregulated, leading to perturbed B cell development, hyperactive B cells, and lethal Ab-mediated autoimmune disease. We have generated a mutant mouse pedigree, termed Mld4, harboring a novel mutation in the gene encoding Lyn, which renders the protein devoid of kinase activity. Despite similarities between the phenotypes of Lyn(Mld4/Mld4) and Lyn(-/-) mice, the spectrum of defects in Lyn(Mld4/Mld4) mice is less severe. In particular, although defects in the B cell compartment are similar, splenomegaly, myeloid expansion, and autoantibody production, characteristic of Lyn(-/-) mice, are absent or mild in Lyn(Mld4/Mld4) mice. Critically, immune complex deposition and complement activation in Lyn(Mld4/Mld4) glomeruli do not result in fulminant glomerulonephritis. Our data suggest that BCR hypersensitivity is insufficient for the development of autoimmune disease in Lyn(-/-) mice and implicate other cell lineages, particularly proinflammatory cells, in autoimmune disease progression. Furthermore, our results provide evidence for an additional role for Lyn kinase, distinct from its catalytic activity, in regulating intracellular signaling pathways.

The process of capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilize an oocyte. A fundamental part of this mechanism is a dramatic increase in the level of tyrosine phosphorylation. Implicated in this process is a unique cAMP/protein kinase A (PKA)-mediated pathway involving an intermediate PKA-activated tyrosine kinase suggested to be pp60(c-src) (SRC) in the mouse. This study has verified the importance of SRC as a key intermediate kinase in promoting the tyrosine phosphorylation events associated with human sperm capacitation. The presence of SRC in human spermatozoa was confirmed immunocytochemically and the kinase was localized to subcellular domains compatible with a role in tyrosine phosphorylation. Additionally SRC co-immunoprecipitated with PKA and became activated by phosphorylation of the Y416 residue during human sperm capacitation. Furthermore, the suppression of PKA and SRC through the application of specific inhibitors led to a dramatic decrease in tyrosine phosphorylation. However, although the inhibition of PKA was also accompanied by a suppression of sperm motility, SRC inhibition did not induce a similar response.

Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.