The protein content of an adaptor protein, STAP-2 is controlled by E3 ubiquitin ligase Cbl

Signal transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2 (SH2)-like domains as well as a YXXQ motif in its C-terminal region. Our previous study in T cells demonstrated that STAP-2 influences FAK protein levels through recruitment of E3 ubiquitin ligase, Cbl, to FAK. In the present study, we found that Cbl directly controls the protein levels and activity of STAP-2. STAP-2 physically interacted with Cbl through its PH and SH2-like domains. Small-interfering RNA-mediated reduction of endogenous Cbl restored STAP-2 protein levels. In contrast, over-expression of Cbl induced STAP-2 degradation. Importantly, Cbl-mediated regulation of STAP-2 protein levels affected Brk/STAP-2-induced STAT3 activation. These results indicate that Cbl regulates STAP-2 protein levels and Brk/STAP-2-mediated STAT3 activation.     

STAP-2/BKS,an adaptor/docking protein,modulates STAT3 activation in acute-phase response through its YXXQ motif

As a c-fms-interacting protein, we cloned a novel adaptor molecule, signal-transducing adaptor protein-2 (STAP-2), which contains pleckstrin homology- and Src homology 2-like (PH and SRC) domains and a proline-rich region. STAP-2 is structurally related to STAP-1/BRDG1 (BCR downstream signaling-1), which we had cloned previously from hematopoietic stem cells. STAP-2 is a murine homologue of a recently identified adaptor molecule, BKS, a substrate of BRK tyrosine kinase. STAP-2 was tyrosine-phosphorylated and translocated to the plasma membrane in response to epidermal growth factor when overexpressed in fibroblastic cells. To define the function of STAP-2, we generated mice lacking the STAP-2 gene. STAP-2 mRNA was strongly induced in the liver in response to lipopolysaccharide and in isolated hepatocytes in response to interleukin-6. In the STAP-2(-/-) hepatocytes, the interleukin-6-induced expression of acute-phase (AP) genes and the tyrosine-phosphorylation level of STAT3 were reduced specifically at the late phase (6-24 h) of the response. These data indicate that STAP-2 plays a regulatory role in the AP response in systemic inflammation. STAP-2 contains a YXXQ motif in the C-terminal region that is a potential STAT3-binding site. Overexpression of wild-type STAP-2, but not of mutants lacking this motif, enhanced the AP response element reporter activity and an AP protein production. These data suggest that STAP-2 is a new class of adaptor molecule that modulates STAT3 activity through its YXXQ motif.     

Regulation of FcepsilonRI-mediated signaling by an adaptor protein STAP-2/BSK in rat basophilic leukemia RBL-2H3 cells

Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma.     

STAP-2 regulates c-Fms/M-CSF receptor signaling in murine macrophage Raw 264.7 cells

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein as a c-Fms/M-CSF receptor-interacting protein and constitutively expressed in macrophages. Our previous studies also revealed that STAP-2 binds to MyD88 and IKK-alpha/beta, and modulates NF-kappaB signaling in macrophages. In the present study, we examined physiological roles of the interaction between STAP-2 and c-Fms in Raw 264.7 macrophage cells. Our immunoprecipitation has revealed that c-Fms directly interacts with the PH domain of STAP-2 independently on M-CSF-stimulation. Ectopic expression of STAP-2 markedly suppressed M-CSF-induced tyrosine phosphorylation of c-Fms as well as activation of Akt and extracellular signal regulated kinase. In addition, Raw 264.7 cells over-expressing STAP-2 showed impaired migration in response to M-CSF and wound-healing process. Taken together, our findings demonstrate that STAP-2 directly binds to c-Fms and interferes with the PI3K signaling, which leads to macrophage motility, in Raw 264.7 cells.     

Modulation of TLR4 signaling by a novel adaptor protein signal-transducing adaptor protein-2 in macrophages

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-kappaB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IkappaB kinase (IKK)-alphabeta, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-alphabeta. These interactions augmented MyD88- and/or IKK-alphabeta-dependent signals, leading to enhancement of the NF-kappaB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-kappaB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.     

Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1(+)c-kit(+)Lin(-)) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1(+)c-kit(+)Lin(-) stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells.     

STAP-2 negatively regulates both canonical and noncanonical NF-kappaB activation induced by Epstein-Barr virus-derived latent membrane protein 1

The signal-transducing adaptor protein 2 (STAP-2) is a recently identified adaptor protein that contains a pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha or IKK-beta and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We found, furthermore, that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with the tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3) and TNFR-associated death domain (TRADD) proteins. STAP-2 mRNA was induced by the expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.     

Signal-transducing adaptor protein-2 regulates integrin-mediated T cell adhesion through protein degradation of focal adhesion kinase

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology- and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, we find that STAP-2-deficient splenocytes or T cells exhibit enhanced cell adhesion to fibronectin after PMA treatment, and that STAP-2-deficient T cells contain the increased protein contents of focal adhesion kinase (FAK). Furthermore, overexpression of STAP-2 induces a dramatic decrease in the protein contents of FAK and integrin-mediated T cell adhesion to fibronectin in Jurkat T cells via the degradation of FAK. Regarding the mechanism for this effect, we found that STAP-2 associates with FAK and enhances its degradation, proteasome inhibitors block FAK degradation, and STAP-2 recruits an endogenous E3 ubiquitin ligase, Cbl, to FAK. These results reveal a novel regulation mechanism for integrin-mediated signaling in T cells via STAP-2, which directly interacts with and degrades FAK.     

Brk activates rac1 and promotes cell migration and invasion by phosphorylating paxillin

Brk (for breast tumor kinase) is a nonreceptor tyrosine kinase containing SH3, SH2, and tyrosine kinase catalytic domains. Brk was originally identified from a human metastatic breast tumor, and its overexpression is frequently observed in breast cancer and several other cancer types. However, the molecular mechanism by which this kinase participates in tumorigenesis remains poorly characterized. In the present study, we not only identified paxillin as the binding partner and substrate of Brk but also discovered a novel signaling pathway by which Brk mediates epidermal growth factor (EGF)-induced paxillin phosphorylation. We show that EGF stimulation activates the catalytic activity of Brk, which in turn phosphorylates paxillin at Y31 and Y118. These phosphorylation events promote the activation of small GTPase Rac1 via the function of CrkII. Through this pathway, Brk is capable of promoting cell motility and invasion and functions as a mediator of EGF-induced migration and invasion. In accordance with these functional roles, Brk translocates to membrane ruffles, where it colocalizes with paxillin during cell migration. Together, our findings identify novel signaling and biological roles of Brk and indicate the first potential link between Brk and metastatic malignancy.     

The BPS domain of Grb10 inhibits the catalytic activity of the insulin and IGF1 receptors

Grb7, Grb10 and Grb14 comprise a family of adaptor proteins that interact with numerous receptor tyrosine kinases upon receptor activation. Between the pleckstrin homology (PH) domain and the Src homology 2 (SH2) domain of these proteins is a region of approximately 50 residues known as the BPS (between PH and SH2) domain. Here we show, using purified recombinant proteins, that the BPS domain of Grb10 directly inhibits substrate phosphorylation by the activated tyrosine kinase domains of the insulin receptor and the insulin-like growth factor 1 (IGF1) receptor. Although inhibition by the BPS domain is dependent on tyrosine phosphorylation of the kinase activation loop, peptide competition experiments indicate that the BPS domain does not bind directly to phosphotyrosine. These studies provide a molecular mechanism by which Grb10 functions as a negative regulator of insulin- and/or IGF1-mediated signaling.     

Regulation of the nonreceptor tyrosine kinase Brk by autophosphorylation and by autoinhibition

Brk (breast tumor kinase) is a nonreceptor tyrosine kinase that is most closely related to the Frk family of kinases, and more distantly to Src family kinases. Brk was originally identified in a screen for tyrosine kinases that are overexpressed in human metastatic breast tumors. To shed light on the activity and regulation of Brk and related tyrosine kinases, we expressed and purified Brk using the Sf9/baculovirus system. We characterized the substrate specificity of Brk using synthetic peptides, and we show that the kinetic parameters K(m) and k(cat) both play a role in specificity. We carried out mass spectrometry experiments to show that Brk autophosphorylates within the predicted kinase activation loop and at additional sites in the N terminus. Autophosphorylation increases enzyme activity of wild-type Brk but not of a Y342A mutant form of Brk. We also carried out experiments to address the possible involvement of the Src homology (SH) 2 and SH3 domains of Brk in enzyme regulation. Mutation of a C-terminal tyrosine (Tyr-447) increases enzyme activity and SH2 domain accessibility, consistent with a role for this residue in autoinhibition. A proline-rich peptide activates Brk, suggesting that the SH3 domain is also involved in maintaining an inactive form of Brk. These biochemical results for Brk may aid in the understanding of other tyrosine kinases in the Frk family.     

Impact of protein tyrosine kinase 6 (PTK6) on human epidermal growth factor receptor (HER) signalling in breast cancer

PTK6, also known as Brk, is highly expressed in over 80% of breast cancers. In the last decade several substrates and interaction partners were identified localising PTK6 downstream of HER receptors. PTK6 seems to be involved in progression of breast tumours, in particular in HER receptor signalling. Here, we show the down-regulation effects of PTK6 in the T47D, BT474 and JIMT-1 breast cancer cell lines. PTK6 knockdown leads to a decreased phosphorylation of HER2, PTEN, MAPK (ERK), p38 MAPK, STAT3 and to a reduced expression of cyclin E. Our findings show that silencing PTK6 impairs the downstream targets of HER receptors and consequently the activation of signalling molecules. Furthermore, lower levels of PTK6 result in reduced migration of T47D and JIMT-1 breast cancer cells. Due to decreased migration, the PTK6 RNA interference might contribute to reduced metastasis and malignant potential of breast cancer cells. Since PTK6 plays an important role in HER receptor signal transduction, its down-regulation might be suitable for future therapy approaches in breast cancer.     

Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines

The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.