Kyung Ko

In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens.

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.

Histidine Phosphorylation of the Potassium Channel KCa3.1 by Nucleoside Diphosphate Kinase B Is Required for Activation of KCa3.1 and CD4 T Cells

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation.

The Phosphatidylinositol 3-Phosphate Phosphatase Myotubularin- Related Protein 6 (MTMR6) Is a Negative Regulator of the Ca2+-Activated K+ Channel KCa3.1

ABSTRACT Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3′ position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P2. MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca2+-activated K channel, KCa3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited KCa3.1 channel activity, and this inhibition required MTMR6s CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit KCa3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited KCa3.1, indicating that MTM CC domains are sufficient to confer target specificity. KCa3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of KCa3.1 by MTMR6 overexpression. These data, when taken together, indicate that KCa3.1 is regulated by PI(3)P and that MTMR6 inhibits KCa3.1 by dephosphorylating the 3′ position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to KCa3.1. KCa3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of KCa3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.

GRIFIN, a Novel Lens-specific Protein Related to the Galectin Family

The vertebrate lens is a relatively simple cellular structure that has evolved to refract light. The ability of the lens to focus light on the retina derives from a number of properties including the expression at high levels of a selection of soluble proteins referred to as the crystallins. In the present study, we have used differential cDNA display techniques to identify a novel, highly abundant and soluble lens protein. Though related to the family of soluble lectins called galectins, it does not bind β-galactoside sugars and has atypical sequences at normally conserved regions of the carbohydrate-binding domain. Like some galectin family members, it can form a stable dimer. It is expressed only in the lens and is located at the interface between lens fiber cells despite the apparent lack of any membrane-targeting motifs. This protein is designated GRIFIN (galectin-relatedinter-fiber protein) to reflect its exclusion from the galectin family given the lack of affinity for β-galactosides. Although the abundance, solubility, and lens-specific expression of GRIFIN would argue that it represents a new crystallin, its location at the fiber cell interface might suggest that its primary function is executed at the membrane.

The B Cell SH2/PH Domain-Containing Adaptor Bam32/DAPP1 Is Required for T Cell-Independent II Antigen Responses

BACKGROUND Bam32/DAPP1 is a B cell adaptor composed of both a PH and an SH2 domain. Previous studies in cell culture and chicken DT40 cells have indicated that Bam32 is critical for normal signaling downstream of the B cell receptor (BCR). RESULTS We now study the function of Bam32 in mice in which Bam32 has been disrupted by a viral gene trap approach. Although B and T cell development is normal in Bam32(-/-) mice, B cell proliferation is reduced by about 50% after BCR crosslinking when compared with Bam32(+/+) mice. Differences in the activation of Erk, Jnk and p38 Map kinases, PLCgamma, and Ca(2+) flux do not account for the defect in proliferation as activation was similar in Bam32(+/+) and Bam32(-/-) B cells. Interestingly, whereas antibody response to T-dependent (TD) and T-independent (TI)-I antigens was similar between Bam32(+/+) and Bam32(-/-) mice, TI-II responses were defective in Bam32(-/-) mice; Bam32(-/-) mice failed to undergo isotype class switch recombination (CSR) to produce IgG3 antibodies due to a cell-autonomous defect in generation of IgG3 germline transcripts. The defect in TI-II antigen response led to an impaired antibody response to immunization with type 3 Streptococcus pneumoniae capsular polyschaccharide (PS), resulting in a markedly increased susceptibility to infection by Streptococcus pneumoniae. CONCLUSIONS These findings indicate that Bam32 specifically couples an upstream signal to the IgG3 isotype heavy chain CSR and suggest that defects in Bam32 may account for the increased susceptibility to encapusulated organisms in a subset of immunodeficient patients.

Phosphatidylinositol-3 Phosphatase Myotubularin-Related Protein 6 Negatively Regulates CD4 T Cells

ABSTRACT Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.

Specificity of the Myotubularin Family of Phosphatidylinositol-3-phosphatase Is Determined by the PH/GRAM Domain

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.

Polycomb Ezh2 controls the fate of GABAergic neurons in the embryonic cerebellum.

Although the genetic interactions between signaling pathways and transcription factors have been largely decoded, much remains to be learned about the epigenetic regulation of cerebellar development. Here, we report that cerebellar deletion of Ezh2, the methyltransferase subunit of the PRC2 complex, results in reduced H3K27me3 and profound transcriptional dysregulation, including that of a set of transcription factors directly involved in cerebellar neuronal cell-type specification and differentiation. Such transcriptional changes lead to increased GABAergic interneurons and decreased Purkinje cells. Transcriptional changes also inhibit the proliferation of granule precursor cells derived from the rhombic lip. The loss of both cell types ultimately results in cerebellar hypoplasia. These findings indicate Ezh2/PRC2 plays crucial roles in regulating neurogenesis from both cerebellar germinal zones. Summary: Cerebellar deletion of Ezh2 leads to increased GABAergic interneurons, decreased Purkinje cells and blocked proliferation of granule precursor cells derived from the rhombic lip.

Epigenetic targeting of bromodomain protein BRD4 counteracts cancer cachexia and prolongs survival

Cancer cachexia is a devastating metabolic syndrome characterized by systemic inflammation and massive muscle and adipose tissue wasting. Although it is responsible for approximately one-third of cancer deaths, no effective therapies are available and the underlying mechanisms have not been fully elucidated. We previously identified the bromodomain and extra-terminal domain (BET) protein BRD4 as an epigenetic regulator of muscle mass. Here we show that the pan-BET inhibitor (+)-JQ1 protects tumor-bearing mice from body weight loss and muscle and adipose tissue wasting. Remarkably, in C26-tumor-bearing mice (+)-JQ1 administration dramatically prolongs survival, without directly affecting tumor growth. By ChIP-seq and ChIP analyses, we unveil that BET proteins directly promote the muscle atrophy program during cachexia. In addition, BET proteins are required to coordinate an IL6-dependent AMPK nuclear signaling pathway converging on FoxO3 transcription factor. Overall, these findings indicate that BET proteins may represent a promising therapeutic target in the management of cancer cachexia.Cachexia is a metabolic syndrome leading to muscle and adipose tissue loss in majority of cancer patients. Here the authors show that, in a mouse model, BET inhibitor JQ1 counteracts muscle and adipose tissue wasting tempering cachexia and prolonging survival through a mechanism unrelated to tumour growth.