The high-affinity IgE receptor (Fc epsilon RI), which is expressed on the surface of mast cells and basophils, has a central role in immediate allergic responses. In the rat basophilic leukaemia cell ...line RBL-2H3, which is a model system for the analysis of Fc epsilon RI-mediated signal transduction, surface engagement of Fc epsilon RI induces histamine release and the tyrosine phosphorylation of several distinct proteins. Although the alpha, beta, and gamma subunits of Fc epsilon RI lack intrinsic tyrosine protein kinase (TPK) activity, a kinase that copurifies with Fc epsilon RI phosphorylates the beta and gamma subunits of the receptor on tyrosine residues. We report here that in RBL-2H3 cells, p56lyn and pp60c-src are activated after Fc epsilon RI crosslinking, and p56lyn coimmunoprecipitates with Fc epsilon RI. In the mouse mast-cell line PT-18, another cell type used to study FC epsilon RI-mediated signalling, tyrosine phosphorylation of proteins is also an immediate consequence of receptor crosslinking. Notably, the only detectable src protein-related TPK in PT-18 cells is p62c-yes, and it is this TPK that is activated on Fc epsilon RI engagement and coimmunoprecipitates with the receptor. Therefore, it seems that different src protein-related TPKs can associate with the same receptor and become activated after receptor engagement.
Coligation of paired immunoglobulin-like receptor B (PIR-B) with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SHP-1 ...and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B; however the molecular target(s) of these phosphatases remain elusive. Here we show that PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igalpha/Igbeta, Syk, Btk and phospholipase C (PLC)-gamma2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-gamma2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-gamma2. Furthermore, we define a requirement for Lyn in mediating tyrosine phosphorylation of PIR-B. Based on these results, we propose a model of PIR-B-mediated inhibitory signaling in which coligation of PIR-B and BCR results in phosphorylation of ITIMs by Lyn, subsequent recruitment of SHP-1, and a resulting inhibition of the BCR-induced inositol 1,4,5-trisphosphate generation by dephosphorylation of Syk and Btk.
Bacterial lipopolysaccharide (LPS) induces a pleiotropic activation of the immune system which might subsequently result in
septic shock. One of the cell surface receptors for LPS is the ...glycophosphatidylinositol-anchored protein CD14. Binding of
LPS to CD14 induces production of lymphokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), IL-6,
and IL-8, and CD14 is subsequently released from the cell surface. However, the mechanism of signaling via CD14 is still not
known. We report here that protein tyrosine kinase (PTK) p56lyn is coupled to the LPS receptor CD14 in human monocytes. LPS
rapidly activates CD14-associated p56lyn simultaneously with PTKs p58hck and p59c-fgr. Inhibition of PTKs by herbimycin A
completely blocks LPS-induced down-modulation of CD14 and production of TNF-alpha and IL-1. These data suggest a critical
role of PTKs in the LPS/CD14-mediated signal transduction pathway in human monocytes.
Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma ...multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.
Activation of platelets with thrombin and other agonists causes a rapid increase in the phosphorylation of multiple proteins on tyrosine. To identify candidate proteintyrosine kinases (PTKs; EC ...2.7.1.112) that may be responsible for these phosphorylation events, we analyzed the expression of seven Src-family PTKs and examined the association of these kinases with known platelet membrane glycoproteins. Five Src-related PTKs were detected in platelets: pp60SRC, pp60FYN, pp62YES, pp61HCK, and two LYN products of Mr54,000 and 58,000. The Fgr and Lck PTKs were not detected. Although strict comparative quantification of protein levels was not possible, pp60SRCwas detected at higher levels than any of the other kinases. In addition, glycoprotein IV (GPIV, CD36), one of the major platelet membrane glycoproteins, was associated in a complex with the Fyn, Yes, and Lyn proteins in platelet lysates. Similar complexes were also found in two GPIV-expressing cell lines, C32 melanoma cells and HEL cells. Since PTKs appear to be involved in stimulus-response coupling at the plasma membrane, these results suggest that ligand interaction with GPIV may activate signaling pathways that are triggered by tyrosine phosphorylation.
A candidate tumor suppressor gene, MMAC1/PTEN, located in human chromosome band 10q23, was recently identified based on sequence alterations observed in several glioma, breast, prostate, and kidney ...tumor specimens or cell lines. To further investigate the mutational profile of this gene in human cancers, we examined a large set of human tumor specimens and cancer cell lines of many types for 10q23 allelic losses and MMAC1 sequence alterations. Loss of heterozygosity (LOH) at the MMAC1 locus was observed in approximately one-half of the samples examined, consistent with the high frequency of 10q allelic loss reported for many cancers. Of 124 tumor specimens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (approximately 10%) of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (approximately 23%). Novel alterations identified in this gene include a missense variant in a melanoma sample and a splicing variant and a nonsense mutation in pediatric glioblastomas. Of 76 tumor cell lines prescreened for probable LOH, microsequence alterations of MMAC1 were detected in 12 (approximately 16%) of the lines, including those derived from astrocytoma, leukemia, and melanoma tumors, as well as bladder, breast, lung, prostate, submaxillary gland, and testis carcinomas. In addition, in this set of tumor cell lines, we detected 11 (approximately 14%) homozygous deletions that eliminated coding portions of MMAC1, a class of abnormality not detected by our methods in primary tumors. These data support the occurrence of inactivating MMAC1 alterations in multiple human cancer types. In addition, we report the discovery of a putative pseudogene of MMAC1 localized on chromosome 9.
Aggregation of Fc gamma RII (CD 32), a low affinity receptor for immunoglobulin G (IgG), on the monocytic cell line THP-1
induces protein tyrosine kinase (PTK) activity. Several distinct cellular ...proteins, including Fc gamma RII itself, are phosphorylated
on tyrosine following cross-linking of the receptor. Fc gamma RII lacks intrinsic PTK activity. In this report we demonstrate
that a kinase activity was coprecipitated with Fc gamma RII in THP-1 cells. The kinetics of the receptor-associated kinase
activity paralleled the appearance of tyrosine phosphorylation events observed following Fc gamma RII activation of THP-1
cells. Several proteins were associated with the receptor. Reimmunoprecipitation analysis demonstrated that lyn gene products
were among the proteins coprecipitated with Fc gamma RII. p59hck (Hck) and p56lyn (Lyn) were the most abundant Src-related
PTKs (Src-PTKs) in THP-1 cells. Enzymatic activity of both kinases, as measured by an in vitro kinase assay, was increased
following specific cross-linking of Fc gamma RII. Furthermore, Fc gamma RII was specifically associated with both enzymes
following its engagement and served as a substrate for both of these kinases. The association of Fc gamma RII with Src-PTK
was specific for Fc gamma RII activation of THP-1 cells, since activation of cells via the high affinity Fc gamma receptor,
Fc gamma RI (CD 64), did not result in association of Fc gamma RII with Hck or Lyn. Our data demonstrate a functional and
physical association of Fc gamma RII with Hck and Lyn consistent with the involvement of Src-PTK in Fc gamma RII-mediated
signal transduction.
Intracellular signal transduction following the extracellular ligation of a
wide variety of different types of surface molecules on leukocytes involves the
activation of protein tyrosine kinases. The ...dependence of successful
intracellular signaling on the functions of the nontransmembrane class of
protein tyrosine kinases coupled with the cell type-specific expression
patterns for several of these enzymes makes them appealing targets for
therapeutic intervention. Development of drugs that can interfere with the
catalytic functions of the nontransmembrane protein tyrosine kinases or that
can disrupt critical interactions with regulatory molecules and/or substrates
should find clinical applications in the treatment of allergic diseases,
autoimmunity, transplantation rejection, and cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such ...as Lck. Most CD45‐deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45‐deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP‐70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45‐deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP‐70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.
CD45 is a tyrosine phosphatase expressed in all hematopoietic cells which is important for signal transduction through the T cell antigen receptor (TCR). Studies using CD45‐deficient cells have ...revealed that Lck, a tyrosine kinase thought to be essential for TCR signaling, is hyperphosphorylated on Y505 in the absence of CD45. This site of tyrosine phosphorylation negatively regulates the function of the Src family of kinases. Here we provide evidence that CD45 can modulate the binding of the Lck to an 11 amino acid tyrosine phosphorylated peptide containing the carboxy‐terminus of Lck (lckP). Significantly, CD45 did not influence the binding of Fyn, PLC gamma 1, GAP and Vav to the same phosphopeptide. Lck protein which bound the peptide was dephosphorylated on Y505 and consisted of only 5–10% of the total cellular Lck. Interestingly, there was a marked increase in binding 15–30 min after CD4 or TCR cross‐linking. Taken together, our data suggest that CD45 specifically modulates the conformation of Lck in a manner consistent with the intramolecular model of regulation of Src‐like kinases.
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