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

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.

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Protein kinase D2 has a restricted but critical role in T-cell antigen receptor signalling in mature T-cells.

PKD (protein kinase D) 2 is a serine/threonine kinase activated by diacylglycerol in response to engagement of antigen receptors in lymphocytes. To explore PKD2 regulation and function in TCR (T-cell antigen receptor) signal transduction we expressed TCR complexes with fixed affinity for self antigens in the T-cells of PKD2-null mice or mice deficient in PKD2 catalytic activity. We also developed a single cell assay to quantify PKD2 activation as T-cells respond to developmental stimuli or engagement of α/ß TCR complexes in vivo. Strikingly, PKD2 loss caused increases in thymic output, lymphadenopathy and splenomegaly in TCR transgenic mice. The precise magnitude and timing of PKD2 activation during T-cell development is thus critical to regulate thymic homoeostasis. PKD2-null T-cells that exit the thymus have a normal transcriptome, but show a limited and abnormal transcriptional response to antigen. Transcriptional profiling reveals the full consequences of PKD2 loss and maps in detail the selective, but critical, function for PKD2 in signalling by α/ß mature TCR complexes in peripheral T-cells.

New insights into the regulation and function of serine/threonine kinases in T lymphocytes.

The development of T lymphocytes in the thymus and the function of mature T cells in adaptive immune responses are choreographed by antigen receptors, costimulatory molecules, adhesion molecules, cytokines, and chemokines. These extrinsic stimuli are coupled to a diverse network of signal transduction pathways that control the transcriptional and metabolic programs that determine T-cell function. At the core of T-lymphocyte signal transduction is the regulated metabolism of inositol phospholipids and the production of two key lipid second messengers: polyunsaturated diacylglycerols (DAGs) and phosphatidylinositol (3-5) triphosphate [PI-(3-5)-P(3)]. The object of the present review is to discuss facts, controversies, and unresolved issues about DAG and PI-(3,4,5)-P(3) production in T lymphocytes and to discuss some of the serine/threonine kinases that control unique aspects of T-lymphocyte biology and coordinate T-cell participation in adaptive immune responses.

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

Mammalian PKD (protein kinase D) isoforms have been implicated in the regulation of diverse biological processes in response to diacylglycerol and PKC (protein kinase C) signalling. To compare the functions of PKD1 and PKD2 in vivo, we generated mice deficient in either PKD1 or PKD2 enzymatic activity, via homozygous expression of PKD1(S744A/S748A) or PKD2(S707A/S711A) 'knockin' alleles. We also examined PKD2-deficient mice generated using 'gene-trap' technology. We demonstrate that, unlike PKD1, PKD2 catalytic activity is dispensable for normal embryogenesis. We also show that PKD2 is the major PKD isoform expressed in lymphoid tissues, but that PKD2 catalytic activity is not essential for the development of mature peripheral T- and B-lymphocytes. PKD2 catalytic activity is, however, required for efficient antigen receptor-induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo. Our results reveal a key in vivo role for PKD2 in regulating the function of mature peripheral lymphocytes during adaptive immune responses. They also confirm the functional importance of PKC-mediated serine phosphorylation of the PKD catalytic domain for PKD activation and downstream signalling and reveal that different PKD family members have unique and non-redundant roles in vivo.

The role of serine/threonine kinases in T-cell activation.

Signalling through serine/threonine kinases is a key mechanism that regulates immune cell development, activation and effector functions. An emerging theme is that serine kinases do not act in isolation, but function in a complex overlapping network. Understanding the molecular targets of serine kinases as well as their links to other serine kinases is key to advancing our understanding of the intracellular signalling pathways that link immune receptors with the gene transcriptional programs that control the immune system in vivo.

Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes.

We have taken a knockout approach to interrogate the function of protein kinase D (PKD) serine/threonine kinases in lymphocytes. DT40 B cells express two PKD family members, PKD1 and PKD3, which are both rapidly activated by the B-cell antigen receptor (BCR). DT40 cells with single or dual deletions of PKD1 and/or PKD3 were viable, allowing the role of individual PKD isoforms in BCR signal transduction to be assessed. One proposed downstream target for PKD1 in lymphocytes is the class II histone deacetylases (HDACs). Regulation of chromatin accessibility via class II histone deacetylases is an important mechanism controlling gene expression patterns, but the molecules that control this key process in B cells are not known. Herein, we show that phosphorylation and nuclear export of the class II histone deacetylases HDAC5 and HDAC7 are rapidly induced following ligation of the BCR or after treatment with phorbol esters (a diacylglycerol mimetic). Loss of either PKD1 or PKD3 had no impact on HDAC phosphorylation, but loss of both PKD1 and PKD3 abrogated antigen receptor-induced class II HDAC5/7 phosphorylation and nuclear export. These studies reveal an essential and redundant role for PKD enzymes in controlling class II HDACs in B lymphocytes and suggest that PKD serine kinases are a critical link between the BCR and epigenetic control of chromatin.

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D.

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.

Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFkappaB activity in vertebrate B-cells.

To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569-1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFkappaB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology.

TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes.

B-lymphocytes possess a specialized lysosomal compartment, the regulated transformation of which has been implicated in B-cell antigen presentation. Members of the mucolipin (TRPML) family of cation channels have been implicated in regulated vesicular transport in several tissues, but a role for TRPML function in lymphocyte vesicular transport physiology has not been previously described. To address the role of TRPML proteins in lymphocyte vesicular transport, we analyzed the lysosomal compartment in cultured B-lymphocytes engineered to lack TRPML1 or after expression of N- or C-terminal GFP fusion proteins of TRPML1 or TRPML2. Consistent with previous analyses of lymphocytes derived from human patients with mutations in TRPML1, we were not able to detect abnormalities in the lysosomes of TRPML1-deficient DT40 B-lymphocytes. However, while N-terminal GFP fusions of TRPML2 localized to normal appearing lysosomes, C-terminal GFP fusions of either TRPML1 or TRPML2 acted to antagonize endogenous TRPML function, localizing to large vesicular structures, the histological properties of which were indistinguishable from the enlarged lysosomes observed in affected tissues of TRPML1-deficient humans. Endocytosed B-cell receptors were delivered to these enlarged lysosomes, demonstrating that a TRPML-dependent process is required for normal regulation of the specialized lysosome compartment of vertebrate B-lymphocytes.

Regulation of protein kinase Cnu by the B-cell antigen receptor.

Diacylglycerol-dependent signaling plays an important role in signal transduction through T- and B-lymphocyte antigen receptors. Recently, a novel serine-threonine kinase of the protein kinase C (PKC) family has been described and designated as PKCnu. PKCnu has two putative diacylglycerol binding C1 domains, suggesting that it may participate in a novel diacylglycerol-mediated signaling pathway. Here we show that both endogenous and recombinant PKCnu are trans-located to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. Mutational analysis demonstrates that PKCnu activation is dependent on trans-phosphorylation of two conserved activation loop serine residues. We also find that PKCnu is an important physiologic target of the B-cell receptor (BCR), because PKCnu is found to be abundantly expressed in chicken and human B-cell lines and, in addition, exhibits robust activation after BCR engagement. Genetic and pharmacologic analyses of BCR-mediated PKCnu activation indicate that it requires intact phospholipase Cgamma and PKC signaling pathways. Furthermore, in co-transfection assays, PKCnu can be trans-phosphorylated by the novel PKC isozymes PKCepsilon, PKCeta, or PKCtheta but not the classical PKC enzyme, PKCalpha. Taken together, these results suggest that PKCnu is an important component of signaling pathways downstream from novel PKC enzymes after B-cell receptor engagement.