Unique functions for protein kinase D1 and protein kinase D2 in mammalian cells

Matthews, Sharon A.; Navarro, M.; Sinclair, Linda V.; Emslie, Elizabeth; Feijoo-Carnero, Carmen; Cantrell, Doreen A.

doi:10.1042/bj20101188

Cited by 76 publications

(165 citation statements)

References 44 publications

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“…PKD2 is the main PKD isotype in human and mouse T lymphocytes. 25,26 We therefore analyzed T lymphoblasts from mice that lacked PKD2 (ref. 26 ) to determine its contribution to MVB traffic and exosome secretion.…”

Section: Resultsmentioning

confidence: 99%

“…25,26 We therefore analyzed T lymphoblasts from mice that lacked PKD2 (ref. 26 ) to determine its contribution to MVB traffic and exosome secretion. T lymphoblasts from WT and PKD2 − / − mice were retrovirally transduced to express GFP-CD63, and then stimulated for 18 h with plate-bound anti-TCR to induce secretory traffic of MVB.…”

Section: Resultsmentioning

confidence: 99%

“…PKD2 is the major PKD isotype expressed in human and mouse T lymphocytes. 25,26 In Jurkat T cells, GFP-tagged PKD1 and endogenous PKD2 have identical regulatory mechanisms, as well as similar activation kinetics and subcellular localization in response to TCR. 62 The effects observed for exogenously expressed PKD1 constructs may thus be the result of inhibition of endogenous PKD2 and activation of PKD2 downstream effectors.…”

Section: Discussionmentioning

confidence: 99%

“…The PKD2 − / − mouse model confirms the contribution of this isoform to exosome secretion, and extends findings from DT40 B and Jurkat T cells to primary T lymphocytes. In response to TCR triggering, PKD2 − / − T lymphoblasts show severe inhibition of IL-2 secretion, 26 although TCR-induced IL-2 gene expression was unaffected. 63 Defective IL-2 secretion by PKD2 − / − T cells and the impaired exosome secretion we describe here could indicate a general secretion defect because of loss of PKD2.…”

Section: Discussionmentioning

confidence: 99%

“…21) are expressed in a wide range of cells, with PKD2 the most abundant isotype in T lymphocytes. 25,26 PKD have two DAG-binding domains (C1a and C1b) at the N terminus, 21 which mediate PKD recruitment to cell membranes. Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation.…”

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confidence: 99%

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Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

et al. 2015

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Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic. Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types. 1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process. 2,3 Two main pathways are involved in MVB maturation. 4,5 In addition to the ESCRT (endosomal complex required for traffic) proteins, 6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA), 7 ceramides 8 and diacylglycerol (DAG) 9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activationinduced cell death (AICD), antigen presentation and intercellular miRNA exchange. [10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,16-21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes. 22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG. 23,24 The secretory vesicle pathway involves several DAGc...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

et al. 2015

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Protein kinase D isoforms are dispensable for integrin‐mediated lymphocyte adhesion and homing to lymphoid tissues

et al. 2012

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Leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4) integrins are essential for lymphocyte adhesion, trafficking and effector functions. Protein kinase D (PKD) has previously been implicated in lymphocyte integrin regulation through regulation of Rap1 activity. However, the true role of PKD in integrin regulation in primary lymphocytes has not previously been investigated. The major PKD isoform in lymphocytes is PKD2. Here we employed PKD2-deficient mice, a specific PKD kinase inhibitor, as well as PKD-null DT40 B cells to investigate the role of PKD in integrin regulation in lymphocytes. We report that PKD2-deficient lymphocytes bound normally to integrin ligands in static and shear flow adhesion assays. They also homed normally to lymphoid organs after adoptive transfer into wild-type mice. DT40 B cells devoid of any PKD isoforms and primary lymphocytes pretreated with a specific PKD inhibitor bound normally to integrin ligands, indicating that multiple PKD isoforms do not redundantly regulate lymphocyte integrins. In addition, PKD2-deficient lymphocytes, as well as DT40 cells devoid of any PKD isoforms, could activate Rap1 in response to B-cell receptor ligation or phorbol ester treatment. Together, these results show that the PKD family does not play a critical role in lymphocyte integrin-mediated cell adhesion or lymphocyte trafficking in vivo.

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A novel Lyn‐protein kinase Cδ/ε‐protein kinase D axis is activated in B cells by signalosome‐independent alternate pathway BCR signaling

Guo

Rothstein

2013

Eur J Immunol

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Summary B cell receptor (BCR) signaling initiates multiple activities critical for B cell function. Recently we identified an alternate BCR signaling pathway, produced by IL-4, that is signalosome-independent, unlike the classical signalosome-dependent pathway, and that leads to activation of the MAP kinase, ERK. In the present study we questioned whether alternate pathway signaling extends to other key downstream events, especially PKD activation. We found that the IL-4-induced alternate pathway for BCR signaling does result in PKD and PKD substrate phosphorylation, and that alternate pathway phosphorylation of HDAC5/7 and other key substrates require PKD. Furthermore, we found that tyrosine phosphorylation of PKCδ/ε occurs as a result of alternate but not classical pathway signaling and is required for phosphorylation of PKD and PKD substrates. This result identifies PKCδ/ε tyrosine phosphorylation as a unique outcome of the alternate pathway. The alternate pathway is mediated by Lyn which is not required for classical pathway signaling and we found that Lyn associates directly with PKCδ/ε and is required for phosphorylation of PKCδ/ε and of PKD. These results indicate that IL-4 influences B cell activation by inducing a novel signaling pathway from BCR to Lyn to PKCδ/ε to PKD.

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Unique functions for protein kinase D1 and protein kinase D2 in mammalian cells

Cited by 76 publications

References 44 publications

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Protein kinase D isoforms are dispensable for integrin‐mediated lymphocyte adhesion and homing to lymphoid tissues

A novel Lyn‐protein kinase Cδ/ε‐protein kinase D axis is activated in B cells by signalosome‐independent alternate pathway BCR signaling

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