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Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-?B activation.

ABSTRACT: Strong NF-?B activation requires ligation of both the CD28 coreceptor and TCR. Phosphoinositide-dependent kinase 1 (PDK1) acts as a scaffold by binding both protein kinase C? (PKC?) and CARMA1, and is therefore essential for signaling to NF-?B. In this article, we demonstrate the importance of PDK1 Thr(513) phosphorylation in regulating the intermolecular organization of PDK1 homodimers. Thr(513) is directly involved in heterotypic PDK1 homodimer formation, in which binding is mediated through the pleckstrin homology (PH) and kinase domains. Upon activation, phosphorylated Thr(513) instead mediates homotypic intermolecular binding through the PH domains. Consequently, cell-permeable peptides with a Thr(513) to Ile derivative (protein transduction domain [PTD]-PDK1-Thr(513)-Ile) bound the kinase domain, whereas a Thr(513)-to-Asp peptide (PTD-PDK1-Thr(513)-Asp) bound the PH domain. PTD-PDK1-Thr(513)-Ile blocked binding between PDK1 and PKC?, phosphorylation of PKC? Thr(538), and activation of both NF-?B and AKT. In contrast, PTD-PDK1- Thr(513)-Asp selectively inhibited binding between PDK1 and CARMA1, and blocked TCR/CD28-induced NF-?B activation. Therefore, Thr(513) phosphorylation regulates a critical intermolecular switch governing PDK1 homodimer structure and the capacity to interact with downstream signaling pathway components. Given the pleiotropic functions of PDK1, these data may open the door to the development of immunosuppressive therapies that selectively target the PDK1 to NF-?B pathway in T cell activation.

SUBMITTER: Kang JA

PROVIDER: S-EPMC3664235 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-κB activation.

Kang Jung-Ah JA Jeong Sang Phil SP Park Daeho D Hayden Matthew S MS Ghosh Sankar S Park Sung-Gyoo SG

Journal of immunology (Baltimore, Md. : 1950) 20130325 9

Strong NF-κB activation requires ligation of both the CD28 coreceptor and TCR. Phosphoinositide-dependent kinase 1 (PDK1) acts as a scaffold by binding both protein kinase Cθ (PKCθ) and CARMA1, and is therefore essential for signaling to NF-κB. In this article, we demonstrate the importance of PDK1 Thr(513) phosphorylation in regulating the intermolecular organization of PDK1 homodimers. Thr(513) is directly involved in heterotypic PDK1 homodimer formation, in which binding is mediated through t ...[more]

PMID: 23530144

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Project description:Hematogenous metastasis involves a glycoprotein mediated adhesion cascade of tumor cells with E-selectin on the endothelial layer of the blood vessels. Cell-cell interactions play a major role in cancer metastasis and invasiveness. Intercellular communication between two cancer cells or between a cancer cell with a stromal cell in the microenvironment such as fibroblasts or inflammatory cells play an important role in metastatic invasion. Culturing tumor cells as 3D spheroids can recapitulate these physiologically relevant cell-cell interactions. The heterogeneity in primary tumors is attributed to cell subpopulations with varying degree of invasiveness. Co-culturing cancer cells with different phenotypes as 3D spheroids can mimic this heterogeneity. Here we report the effect of homotypic and heterotypic interactions in breast cancer cells cultured as 3D spheroids on polydimethylsiloxane (PDMS) on the adhesion phenotype to E-selectin. We show that breast cancer cell lines (BT20 and MCF7) propagating as 3D spheroids on PDMS exhibit a stronger interaction with human recombinant E-selectin when compared to their respective monolayer grown counterparts on tissue culture plate (TCP). Matrigel invasion assay also indicated that BT20 and MCF7 spheroids were more invasive than BT20 and MCF7 cells grown as monolayers. To mimic tumor heterogeneity in vitro, a co-culture model included tumorigenic cell lines BT20, MCF7 and a non-tumorigenic mammary epithelial cell line MCF10A. These cell lines were cultured together in equal seeding ratio on PDMS to generate co-culture spheroids. The heterotypic interactions in the co-culture model resulted in enhancement of the adhesion of the most invasive BT20 cell line to E-selectin. BT20 cells in co-culture bound to the greatest degree to soluble E-selectin compared to MCF7 and MCF10A cells in co-culture. Co-invasion assay with co-culture spheroids indicated that BT20 cells in co-culture were more invasive than MCF7 and MCF10A cells. The results presented here indicate that homotypic and heterotypic interaction of cancer cells favor adhesion to E-selectin thus representing a complexity beyond planar cell culture. Also, when cells of different phenotypes are mixed, the heterogeneity enhances the adhesive phenotype and invasiveness of the most invasive cell population. The results challenge the classic use of planar cell culture for evaluating the adhesion of cancer cells to E-selectin and establish our co-culture technique as a model that can help investigative studies in metastasis and invasiveness of breast and other types of cancers.

Dataset Information

Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-?B activation.

Publications

Transition from heterotypic to homotypic PDK1 homodimerization is essential for TCR-mediated NF-κB activation.

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