A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

Bartolo, Vincenzo Di; Montagne, Benjamin; Salek, Mogjiborahman; Jungwirth, Britta; Carrette, Florent; Fourtane, Julien; Sol‐Foulon, Nathalie; Michel, Frédérique; Schwartz, Olivier; Lehmann, Wolf D.; Acuto, Oreste

doi:10.1084/jem.20062066

Cited by 91 publications

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“…Unexpectedly, and as yet poorly understood, HPK1 negatively regulates TCR signaling [12,19,22,23]. Inhibition of NFkB through the HPK1 C-terminus in apoptotic cells and recruitment of inhibitory molecules to the HPK1 phosphorylation site on SLP-76 have been reported [14,19,22].…”

Section: Introductionmentioning

confidence: 99%

“…Following recruitment to the TCR, HPK1 is phosphorylated on tyrosine 379 and binds the SH2 domain of SLP-76, which itself is phosphorylated by HPK1 [15][16][17][18][19]. Subsequent transphosphorylation by PKD1 and autophosphorylation within the kinase domain result in full activation of HPK1 [15], which then regulates different cellular responses including apoptosis, activationinduced cell death and autoimmunity [20][21][22].Unexpectedly, and as yet poorly understood, HPK1 negatively regulates TCR signaling [12,19,22,23]. Inhibition of NFkB through the HPK1 C-terminus in apoptotic cells and recruitment of inhibitory molecules to the HPK1 phosphorylation site on SLP-76 have been reported [14,19,22].…”

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confidence: 99%

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“…Inhibition of NFkB through the HPK1 C-terminus in apoptotic cells and recruitment of inhibitory molecules to the HPK1 phosphorylation site on SLP-76 have been reported [14,19,22]. We wondered if HPK1 functionally competes with ADAP for SLP-76 binding and investigated a possible role of HPK1 in T-cell adhesion and LFA-1 activation.…”

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HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

et al. 2010

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The hematopoietic progenitor kinase 1 (HPK1) signals into MAPK and NFjB pathways downstream of immunoreceptors, but enigmatically is a negative regulator of leukocytes. Here, we report a novel role for HPK1 in regulating the activation of the adhesion molecule leukocyte function-associated antigen-1 (LFA-1). Upon TCR stimulation, mediated by binding of adhesion and degranulation promoting adaptor protein (ADAP) to SLP-76, a ternary complex composed of ADAP/55-kDa src kinase associated phosphoprotein (SKAP-55) and RIAM translocates to the membrane and causes membrane recruitment of the active small GTPase Ras-related protein 1 (Rap1). Active Rap1, via its binding to RapL (regulator for cell adhesion and polarization enriched in lymphoid tissues), mediates LFA-1 integrin activation. We show here that HPK1, which also binds SLP-76, compete with ADAP for SLP-76 binding. In addition, HPK1 dampens Rap1 activation, resulting in decreased LFA-1 activity. Analysis of HPK1-deficient T cells revealed increased ADAP recruitment to SLP-76 and elevated Rap1 activation in those cells, leading to increased adhesion to ICAM-1 and cell spreading. Altogether, these results describe a novel function for HPK1 in linking TCR signaling to cell adhesion regulation and provide a mechanistic explanation for the negative regulatory role of HPK1 in T-cell biology.Key words: ADAP . HPK1 . LFA-1 . Rap1 . SLP-76Supporting Information available online Introduction T-cell adhesion to other immune cells or endothelium is crucial in generating an immune response. Central mediators of T-cell adhesion are integrins as, for example, the aLb2 integrin leukocyte function-associated antigen-1 (LFA-1), whose activation is mediated by chemokines or antigen receptor encounter and the b1 family of integrins (very late antigen; a4b1, a5b1 and a6b1). LFA-1 activation after TCR stimulation, also referred to as inside-out signaling, relies on a signaling module composed of the transmembranous adapter LAT (linker for activation of T cells) and the cytosolic adaptors SLP-76 (76-kDa src homology 2 domain-containing leukocyte phosphoprotein) and Gads (Grb2-related adaptor downstream of Shc) [1,2]. Two additional adapter molecules, adhesion and degranulation promoting adaptor protein (ADAP) and 55-kDa src kinase associated phosphoprotein 3220ing adapter molecule) which binds active Rap1 and thereby facilitates TCR-mediated integrin activation [5,6]. In addition, the Rap1-GTP effector regulator for cell adhesion and polarization enriched in lymphoid tissues (RapL) was recently shown to directly bind SKAP-55, which might differentially influence integrin affinity/avidity regulation [7]. Additional effectors of active Rap1 involved in integrin activation include the kinase Mst1 downstream of RapL and the serine/threonine kinase PKD1 [8][9][10].The hematopoietic progenitor kinase 1 (HPK1), a Ste20-homologous serine/threonine kinase, is activated upon TCR stimulation and feeds into MAPK and NFkB signaling [11][12][13][14]. Following recruitment to the TCR, HPK1 is pho...

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HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

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“…A single SH2 domain resides in the C-terminal portion of SLP-76 and links it to the adhesion and degranulation-promoting adaptor protein (ADAP) and to the hematopoietic progenitor kinase 1 [17][18][19]. Hematopoietic progenitor kinase 1, in turn, has been shown to phosphorylate SLP-76, creating a binding site for 14-3-3, a negative regulator of TCR signaling [20,21]. The central proline-rich region binds phospholipase Cc-1 [22] and the Src family kinase lck [23] and also contains a novel SH3 domain binding motif (RxxK) that is responsible for the constitutive association of SLP-76 with Gads [24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

Jordan¹,

Kliche²,

Shabason³

et al. 2007

Eur J Immunol

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Multi-molecular complexes nucleated by adaptor proteins play a central role in signal transduction. In T cells, one central axis consists of the assembly of several signaling proteins linked together by the adaptors linker of activated T cells (LAT), Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads). Each of these adaptors has been shown to be important for normal T cell development, and their proper sub-cellular localization is critical for optimal function in cell lines. We previously demonstrated in Jurkat T cells and a rat basophilic leukemic cell line that expression of a 50-amino acid polypeptide identical to the site on SLP-76 that binds to Gads blocks proper localization of SLP-76 and SLP-76-dependent signaling events. Here we extend these studies to investigate the ability of this polypeptide to inhibit TCR-induced integrin activity in Jurkat cells and to inhibit in vivo thymocyte development and primary T cell function. These data provide evidence for the in vivo function of a dominant-negative peptide based upon the biology of SLP-76 action and suggest the possibility of therapeutic potential of targeting the SLP-76/Gads interaction. IntroductionStimulation of T cells through the T cell receptor (TCR) leads to recruitment and localization of several critical signaling proteins, which results in the generation of a macro-molecular signaling complex. Key components of this complex, or signalosome, include the adaptors Src homology 2 (SH2) domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads), which serves to link SLP-76 and its associated molecules to the transmembrane adaptor linker of activated T cells (LAT) [1].As an adaptor protein, SLP-76 possesses no enzymatic activity but functions via interaction with multiple proteins by way of three structural domains [2][3][4]. The N terminus harbors three tyrosines that become phos- phorylated upon TCR engagement [5,6] and have been reported to serve as docking sites for the guanine nucleotide exchange factor Vav, the adaptor protein Nck, the Tec family kinase Itk, and the p85 subunit of PI3K [7][8][9][10][11][12][13][14][15][16]. A single SH2 domain resides in the C-terminal portion of SLP-76 and links it to the adhesion and degranulation-promoting adaptor protein (ADAP) and to the hematopoietic progenitor kinase 1 [17][18][19]. Hematopoietic progenitor kinase 1, in turn, has been shown to phosphorylate SLP-76, creating a binding site for 14-3-3, a negative regulator of TCR signaling [20,21]. The central proline-rich region binds phospholipase Cc- Targeted deletion of SLP-76 in mice results in an inability of thymocytes to progress past the CD4 -CD8 -double-negative (DN) stage of development and thus a complete absence of mature T cells [28,29]. Since T cells do not develop in the absence of SLP-76, much of our knowledge of the role of SLP-76 in mature TCR signaling has been derived from the study of J14 ...

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The 14‐3‐3/SLP76 protein–protein interaction in T‐cell receptor signalling: a structural and biophysical characterization

et al. 2020

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The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3r. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling.

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A novel pathway down-modulating T cell activation involves HPK-1–dependent recruitment of 14-3-3 proteins on SLP-76

Cited by 91 publications

References 47 publications

HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

HPK1 competes with ADAP for SLP‐76 binding and via Rap1 negatively affects T‐cell adhesion

In vivo disruption of T cell development by expression of a dominant‐negative polypeptide designed to abolish the SLP‐76/Gads interaction

The 14‐3‐3/SLP76 protein–protein interaction in T‐cell receptor signalling: a structural and biophysical characterization

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