Brandon J. Burbach

The syndecan-1 ectodomain regulates αvβ3 integrin activity in human mammary carcinoma cells

The αvβ3 integrin participates in cell morphogenesis, growth factor signaling, and cell survival. Activation of the integrin is central to these processes and is influenced by specific ECM components, which engage both integrins and syndecans. This paper demonstrates that the αvβ3 integrin and syndecan-1 (S1) are functionally coupled. The integrin is dependent on the syndecan to become activated and to mediate signals required for MDA-MB-231 and MDA-MB-435 human mammary carcinoma cell spreading on vitronectin or S1-specific antibody. Coupling of the syndecan to αvβ3 requires the S1 ectodomain (ED), as ectopic expression of glycosylphosphatidylinositol-linked S1ED enhances αvβ3 recognition of vitronectin; and treatments that target this domain, including competition with recombinant S1ED protein or anti-S1ED antibodies, mutation of the S1ED, or down-regulation of S1 expression by small-interfering RNAs, disrupt αvβ3-dependent cell spreading and migration. Thus, S1 is likely to be a critical regulator of many cellular behaviors that depend on activated αvβ3 integrins.

β-Actin specifically controls cell growth, migration, and the G-actin pool

Targeted deletion of Actb demonstrates that the β-actin gene, in contrast to the γ-actin gene, is an essential gene uniquely required for cell growth and migration. Cell motility and growth defects in β-actin–knockout primary cells are due to a specific role for β-actin in regulating gene expression through control of the cellular G-actin pool.

T-cell receptor signaling to integrins.

Summary:  Integrin adhesion receptors are critical for antigen recognition by T cells and for regulated recirculation and trafficking into and through various tissues in the body. T‐cell receptor (TCR) signaling induces rapid increases in integrin function that facilitate T‐cell activation by promoting stable contact with antigen‐presenting cells and extracellular proteins in the environment. In this review, we outline the molecular mechanisms by which the TCR signals to integrins and present a model that highlights four key events: (i) initiation of proximal TCR signals nucleated by the linker for activated T cells (LAT) adapter protein and involving Itk, phospholipase C‐γ1, Vav1, and Src homology 2 domain‐containing leukocyte‐specific phosphoprotein of 76 kDa; (ii) transmission of integrin activation signals from the LAT signalosome to integrins by protein kinase (PK) C and the adapter protein, adhesion and degranulation‐promoting adapter protein; (iii) assembly of integrin‐associated signaling complexes that include PKD, the guanosine triphosphatase Rap1 and its effectors, and talin; and (iv) reorganization of the actin cytoskeleton by WAVE2 and other actin‐remodeling proteins. These events coordinate changes in integrin conformation and clustering that result in enhanced integrin functional activity following TCR stimulation.

Syndecan-1 regulates αvβ5 integrin activity in B82L fibroblasts

B82L mouse fibroblasts respond to fibronectin or vitronectin via a syndecan-1-mediated activation of the αvβ5 integrin. Cells attached to syndecan-1-specific antibody display only filopodial extension. However, the syndecan-anchored cells extend lamellipodia when the antibody-substratum is supplemented with serum, or low concentrations of adsorbed vitronectin or fibronectin, that are not sufficient to activate the integrin when plated alone. Integrin activation is blocked by treatment with (Arg-Gly-Asp)-containing peptides and function-blocking antibodies that target αv integrins, as well as by siRNA-mediated silencing of β5 integrin expression. In addition, αvβ5-mediated cell attachment and spreading on high concentrations of vitronectin is blocked by competition with recombinant syndecan-1 ectodomain core protein and by downregulation of mouse syndecan-1 expression by mouse-specific siRNA. Taking advantage of the species-specificity of the siRNA, rescue experiments in which human syndecan-1 constructs are expressed trace the activation site to the syndecan-1 ectodomain. Moreover, both full-length mouse and human syndecan-1 co-immunoprecipitate with the β5 integrin subunit, but fail to do so if the syndecan is displaced by competition with soluble, recombinant syndecan-1 ectodomain. These results suggest that the ectodomain of the syndecan-1 core protein contains an active site that assembles into a complex with the αvβ5 integrin and regulates αvβ5 integrin activity.

Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells

Expression patterns of syndecan-1, the cell surface heparan sulfate proteoglycan (HSPG) predominant on epithelial cells, were analyzed in tissue samples from 30 infiltrating human breast carcinomas and in 9 human breast carcinoma cell lines. Immunohistochemical staining demonstrates that while a subset of the breast carcinomas lose syndecan-1, this proteoglycan is expressed or overexpressed in a majority of the cases. Interestingly, cells in poor grade tumors contain intracellular syndecan-1, an observation that has not been previously described and was thus further investigated. Examination of cultured breast carcinoma cell lines indicates that they also display the phenotype of the syndecan-1 positive tumors and thereby provide a model system for analysis of intracellular syndecan-1. All cell lines examined express syndecan-1, and poorly differentiated lines such as BT549 cells internalize the proteoglycan from the cell surface where it accumulates as intact HSPG in intracellular vesicles. Colocalization studies using fluorescent markers identify these to be lysosomes. This finding is unexpected, as the accepted mechanism for degradation of syndecan HSPG following endocytosis is fragmentation of the protein core and glycosaminoglycan chains in endosomes, followed by delivery of the fragments to lysosomes. Lysosomal inactivation using ammonium chloride demonstrates that well-differentiated lines such as T47D and MCF-7 cells, which maintain the majority of syndecan-1 on their cell surfaces, also target intact constitutively endocytosed syndecan-1 to lysosomes. Taken together, these results suggest that mammary epithelial cells utilize a previously uncharacterized mechanism for syndecan-1 catabolism. In this pathway the proteoglycan remains intact as it passes through the endosomal system, prior to arriving at its site of intracellular degradation in lysosomes.

Adhesion and Degranulation-Promoting Adapter Protein (ADAP) Positively Regulates T Cell Sensitivity to Antigen and T Cell Survival

The hemopoietic specific adapter protein ADAP (adhesion and degranulation-promoting adapter protein) positively regulates TCR-dependent, integrin-mediated adhesion and participates in signaling pathways downstream of the TCR that result in T cell activation. The specific role of ADAP in regulating Ag-dependent T cell interactions with APCs and T cell activation following Ag stimulation is not known. We used ADAP−/− DO11.10 T cells to demonstrate that ADAP promotes T cell conjugation to Ag-laden APCs. Complementary in vitro and in vivo approaches reveal that ADAP controls optimal T cell proliferation, cytokine production, and expression of the prosurvival protein Bcl-xL in response to limiting Ag doses. Furthermore, ADAP is critical for clonal expansion in vivo independent of Ag concentration under conditions of low clonal abundance. These results suggest that ADAP regulates T cell activation by promoting Ag-dependent T cell-APC interactions, resulting in enhanced T cell sensitivity to Ag, and by participating in prosurvival signaling pathways initiated by Ag stimulation.

NF-κB Activation in T Cells Requires Discrete Control of IκB Kinase α/β (IKKα/β) Phosphorylation and IKKγ Ubiquitination by the ADAP Adapter Protein

NF-κB activation following engagement of the antigen-specific T cell receptor involves protein kinase C-θ-dependent assembly of the CARMA1-BCL10-MALT1 (CBM) signalosome, which coordinates downstream activation of IκB kinase (IKK). We previously identified a novel role for the adhesion- and degranulation-promoting adapter protein (ADAP) in regulating the assembly of the CBM complex via an interaction of ADAP with CARMA1. In this study, we identify a novel site in ADAP that is critical for association with the TAK1 kinase. ADAP is critical for recruitment of TAK1 and the CBM complex, but not IKK, to protein kinase C-θ. ADAP is not required for TAK1 activation. Although both the TAK1 and the CARMA1 binding sites in ADAP are essential for IκBα phosphorylation and degradation and NF-κB nuclear translocation, only the TAK1 binding site in ADAP is necessary for IKK phosphorylation. In contrast, only the CARMA1 binding site in ADAP is required for ubiquitination of IKKγ. Thus, distinct sites within ADAP control two key activation responses that are required for NF-κB activation in T cells.

Intravital mucosal imaging of CD8 + resident memory T cells shows tissue-autonomous recall responses that amplify secondary memory

CD8+ T cell immunosurveillance dynamics influence the outcome of intracellular infections and cancer. Here we used two-photon intravital microscopy to visualize the responses of CD8+ resident memory T cells (TRM cells) within the reproductive tracts of live female mice. We found that mucosal TRM cells were highly motile, but paused and underwent in situ division after local antigen challenge. TRM cell reactivation triggered the recruitment of recirculating memory T cells that underwent antigen-independent TRM cell differentiation in situ. However, the proliferation of pre-existing TRM cells dominated the local mucosal recall response and contributed most substantially to the boosted secondary TRM cell population. We observed similar results in skin. Thus, TRM cells can autonomously regulate the expansion of local immunosurveillance independently of central memory or proliferation in lymphoid tissue.Masopust and colleagues show that mucosal tissue-resident memory T cells proliferate in situ in response to local antigen and dominate the local recall response.

Distinct Regulation of Integrin-Dependent T Cell Conjugate Formation and NF-κB Activation by the Adapter Protein ADAP

Following TCR stimulation, T cells utilize the hematopoietic specific adhesion and degranulation-promoting adapter protein (ADAP) to control both integrin adhesive function and NF-κB transcription factor activation. We have investigated the molecular basis by which ADAP controls these events in primary murine ADAP−/− T cells. Naive DO11.10/ADAP−/− T cells show impaired adhesion to OVAp (OVA aa 323–339)-bearing APCs that is restored following reconstitution with wild-type ADAP. Mutational analysis demonstrates that the central proline-rich domain and the C-terminal domain of ADAP are required for rescue of T:APC conjugate formation. The ADAP proline-rich domain is sufficient to bind and stabilize the expression of SKAP55 (Src kinase-associated phosphoprotein of 55 kDa), which is otherwise absent from ADAP−/− T cells. Interestingly, forced expression of SKAP55 in the absence of ADAP is insufficient to drive T:APC conjugate formation, demonstrating that both ADAP and SKAP55 are required for optimal LFA-1 function. Additionally, the ADAP proline-rich domain is required for optimal Ag-induced activation of CD69, CD25, and Bcl-xL, but is not required for assembly of the CARMA1/Bcl10/Malt1 (caspase-recruitment domain (CARD) membrane-associated guanylate kinase (MAGUK) protein 1/B-cell CLL-lymphoma 10/mucosa-associated lymphoid tissue lymphoma translocation protein 1) signaling complex and subsequent TCR-dependent NF-κB activity. Our results indicate that ADAP is used downstream of TCR engagement to delineate two distinct molecular programs in which the ADAP/SKAP55 module is required for control of T:APC conjugate formation and functions independently of ADAP/CARMA1-mediated NF-κB activation.

The Pleckstrin Homology Domain in the SKAP55 Adapter Protein Defines the Ability of the Adapter Protein ADAP To Regulate Integrin Function and NF-κB Activation

Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter Src kinase-associated phosphoprotein of 55 kDa (SKAP55). In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP–ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP–ADAP chimera associated with the LFA-1 integrin after TCR stimulation. Although the SKAP–ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP−/− T cells. A single point mutation in the pleckstrin homology domain of SKAP55 (R131M) blocks the ability of the SKAP–ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.