PKC-θ selectively controls the adhesion-stimulating molecule Rap1

Letschka, Thomas; Kollmann, Veronika; Pfeifhofer-Obermair, Christa; Lutz-Nicoladoni, Christina; Obermair, Gerald J.; Fresser, Friedrich; Leitges, Michael; Hermann-Kleiter, Natascha; Kaminski, Sandra; Baier, Gottfried

doi:10.1182/blood-2007-11-121111

Cited by 58 publications

(49 citation statements)

References 68 publications

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“…Interestingly, CRK/CRKL Dko T cells chemotaxed efficiently toward both CCL19 and CCL21 in this setting ( Figure 3D and Table 1). Comparison of multiple parameters including velocity, directionality, and forward migration index showed no significant differences between mutant and pathway (16). Similar adhesion defects were observed in response to fibronectin, a ligand for the β 1 integrin VLA-4 ( Figure 2B).…”

Section: Introductionsupporting

confidence: 53%

“…For example, Ccr7 -/-T cells show defects in trafficking to recipient lymph nodes, while Cd18 -/-T cells migrate efficiently to lymph nodes but are defective in their ability to enter inflamed skin and GVHD target tissues such as intestine (50,53). PKCθ deficiency was shown to affect GVHD via altered T cell effector function (52), but trafficking may also be affected, since PKCθ is known to regulate RAP1 activation and T cell trafficking in other settings (16,54). CRK-deficient T cells, which migrate efficiently to lymphoid organs but inefficiently to liver in a GVHD model, most closely resemble the Cd18 -/-T cells.…”

Section: Methodsmentioning

confidence: 99%

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CRK proteins selectively regulate T cell migration into inflamed tissues

Huang¹,

Clarke²,

Karimi³

et al. 2015

J. Clin. Invest.

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R e s e a R c h a R t i c l e1 0 2 0 jci.org Volume 125 Number 3 March 2015 therapeutic implications, since they can carry out graftversus-leukemia (GVL) responses with minimal GVHD. Results Generation and characterization of T cell-specific CRK T cells (data not shown).Western blotting of purified CD4 + T cells from Dko and WT mice revealed that levels of CRKI, CRKII, and CRKL in the mutant T cells were reduced by at least 95% ( Figure 1A and data not shown), and flow cytometric analysis confirmed loss of CRK protein expression (Figure 1, B and C).CRK/CRKL Dko mice were born at Mendelian ratios, and spleen, thymus, and lymph nodes exhibited normal cellularity (data not shown). Thymocyte populations were similar in WT and Dko mice, indicating that T cell development proceeded normally ( Figure 1D). Peripheral lymphoid organs showed no differences in proportions of CD4 + and CD8 + T cells, naive (CD62L hi CD44 lo ), memory (CD62L lo CD44 hi ), or activated (CD69 hi ) T cell subsets (Figure 1, D and E, and data not shown). Thus, these mice represented a suitable source of mature CRK/CRKL Dko T cells for functional studies. CRK/CRKL-deficient T cells show defects in adhesion and polarization.Since CRK proteins control adhesion in non-hematopoietic cells (4), we first asked whether integrin-dependent adhesion is defective in CRK/CRKL Dko T cells. On plates coated with ICAM-1, the ligand for the β 2 integrin LFA-1, WT preactivated CD4 + T cells showed about 10% basal binding, which was increased 2-to 5-fold after stimulation with the chemokines CXCL12 or CCL21, or anti-CD3 (Figure 2A). CRK/CRKL Dko T cells showed a significant reduction in adhesion to ICAM-1 under both basal and stimulated conditions. Indeed, chemokine stimulation induced almost no increased adhesion in these cells. Treatment with PMA bypassed the defect. This is most likely because the defect in CRK/CRKL Dko cells lies upstream of PKC signaling in the pathway leading to integrin activation, though PKC activation could also drive a parallel

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Section: Introductionsupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

CRK proteins selectively regulate T cell migration into inflamed tissues

Huang¹,

Clarke²,

Karimi³

et al. 2015

J. Clin. Invest.

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“…Its presence in neutrophils was demonstrated in 2002 (23), but its function was not investigated. In T cells, PKC-u has been shown to mediate the activation of LFA-1 after TCR stimulation (27). This signaling cascade shares some similarities with the insideout signaling pathway following the activation of neutrophil GPCRs by chemokines.…”

Section: Discussionmentioning

confidence: 87%

“…Recently, PKC-u has been shown to regulate the affinity of the b 2 integrin LFA-1 downstream of the TCR by phosphorylating the guanine nucleotide exchange factor Rap-GEF2, an activator of the small GTPase Rap1. This leads to a stable immunological synapse (27). The signaling cascade leading to LFA-1 activation after TCR stimulation shares some similarities with the inside-out signaling pathway following the activation of neutrophil GPCRs by chemokines.…”

mentioning

confidence: 97%

Protein Kinase C-θ Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow

Bertram

Zhang

Vietinghoff

et al. 2012

The Journal of Immunology

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Protein kinase C (PKC)-θ is involved in T cell activation via regulating the avidity of the β2 integrin LFA-1 in the immunological synapse. LFA-1 also mediates leukocyte adhesion. To investigate the role of PKC-θ in neutrophil adhesion, we performed intravital microscopy in cremaster venules of mice reconstituted with bone marrow from LysM-GFP+ (wild-type [WT]) and PKC-θ gene-deficient (Prkcq−/−) mice. Following stimulation with CXCL1, both WT and Prkcq−/− cells became adherent. Although most WT neutrophils remained adherent for at least 180 s, 50% of Prkcq−/− neutrophils were detached after 105 s and most by 180 s. Upon CXCL1 injection, rolling of all WT neutrophils stopped for 90 s, but rolling of Prkcq−/− neutrophils started 30 s after CXCL1 stimulation. A similar neutrophil adhesion defect was seen in vitro, and spreading of Prkcq−/− neutrophils was delayed. Prkcq−/− neutrophil recruitment was impaired in fMLP-induced transmigration into the cremaster muscle, thioglycollate-induced peritonitis, and LPS-induced lung injury. We conclude that PKC-θ mediates integrin-dependent neutrophil functions and is required to sustain neutrophil adhesion in postcapillary venules in vivo. These findings suggest that the role of PKC-θ in outside–in signaling following engagement of neutrophil integrins is relevant for inflammation in vivo.

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“…By phosphorylation of SLP-76 HPK1 provides a docking site for 14-3-3, which can undergo an inhibitory interaction with PKCy. In HPK1 À/À T cells, enhanced PKCy activity, a positive regulator of Rap1 [28], might contribute to the elevated Rap1 activity. In addition, HPK1 has the potential to dampen Rap1 by sequestration of C3G via the HPK1 interacting protein CrkL.…”

Section: Discussionmentioning

confidence: 99%

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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PKC-θ selectively controls the adhesion-stimulating molecule Rap1

Cited by 58 publications

References 68 publications

CRK proteins selectively regulate T cell migration into inflamed tissues

CRK proteins selectively regulate T cell migration into inflamed tissues

Protein Kinase C-θ Is Required for Murine Neutrophil Recruitment and Adhesion Strengthening under Flow

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

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