Zhai Li

Identification and analysis of PH domain-containing targets of phosphatidylinositol 3-kinase using a novel in vivo assay in yeast.

Phosphatidylinositol 3‐kinase (PI3K) mediates a variety of cellular responses by generating PtdIns(3,4)P2 and PtdIns(3,4,5)P3. These 3‐phosphoinositides then function directly as second messengers to activate downstream signaling molecules by binding pleckstrin homology (PH) domains in these signaling molecules. We have established a novel assay in the yeast Saccharomyces cerevisiae to identify proteins that bind PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in vivo which we have called TOPIS (Targets of PI3K Identification System). The assay uses a plasma membrane‐targeted Ras to complement a temperature‐sensitive CDC25 Ras exchange factor in yeast. Coexpression of PI3K and a fusion protein of activated Ras joined to a PH domain known to bind PtdIns(3,4)P2 (AKT) or PtdIns(3,4,5)P3 (BTK) rescues yeast growth at the non‐permissive temperature of 37°C. Using this assay, we have identified several amino acids in the β1–β2 region of PH domains that are critical for high affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have proposed a structural model for how these PH domains might bind PI3K products with high affinity. From these data, we derived a consensus sequence which predicts high‐affinity binding to PtdIns(3,4)P2 and/or PtdIns(3,4,5)P3, and we have identified several new PH domain‐containing proteins that bind PI3K products, including Gab1, Dos, myosinX, and Sbf1. Use of this assay to screen for novel cDNAs which rescue yeast at the non‐permissive temperature should provide a powerful approach for uncovering additional targets of PI3K.

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.

Inhibition of the K+ channel KCa3.1 ameliorates T cell–mediated colitis

The calcium-activated K+ channel KCa3.1 plays an important role in T lymphocyte Ca2+ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca2+ influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1−/− mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1−/− mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor–stimulated Ca2+ influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca2+ influx and cytokine production were impaired in KCa3.1−/− Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1−/− protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2−/− recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis.

Protein histidine phosphatase 1 negatively regulates CD4 T cells by inhibiting the K+ channel KCa3.1

The calcium activated K+ channel KCa3.1 plays an important role in T lymphocyte Ca2+ signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca2+ influx. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, is required for KCa3.1 channel activation in human CD4 T lymphocytes. We now show that the mammalian protein histidine phosphatase (PHPT-1) directly binds and inhibits KCa3.1 by dephosphorylating histidine 358 on KCa3.1. Overexpression of wild-type, but not a phosphatase dead, PHPT-1 inhibited KCa3.1 channel activity. Decreased expression of PHPT-1 by siRNA in human CD4 T cells resulted in an increase in KCa3.1 channel activity and increased Ca2+ influx and proliferation after T cell receptor (TCR) activation, indicating that endogenous PHPT-1 functions to negatively regulate CD4 T cells. Our findings provide a previously unrecognized example of a mammalian histidine phosphatase negatively regulating TCR signaling and are one of the few examples of histidine phosphorylation/dephosphorylation influencing a biological process in mammals.

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.

Genetic Analysis of the Myotubularin Family of Phosphatases in Caenorhabditis elegans

Myotubularins (MTMs) constitute a large family of lipid phosphatases that specifically dephosphorylate phosphatidylinositol (3)P. MTM1 and MTM2 are mutated in X-linked myotubular myopathy and Charcot-Marie-Tooth disease (type 4B), respectively, although the mechanisms whereby MTM dysfunction leads to these diseases is unknown. To gain insight into MTM function, we undertook the study of MTMs in the nematode Caenorhabditis elegans, which possesses representative homologues of the four major subgroups of MTMs identified in mammals. As in mammals, we found that C. elegans MTMs mediate distinct functions. let-512 (vps34) encodes the C. elegans homologue of the yeast and mammalian homologue of the phosphatidylinositol 3-kinase Vps34. We found that reduction of mtm-6 (F53A2.8) function by RNA inhibition rescued the larval lethality of let-512 (vps34) mutants and that the reduction of mtm-1 (Y110A7A.5) activity by RNA inhibition rescued the endocytosis defect of let-512 animals. Together, these observations provide genetic evidence that MTMs negatively regulate phosphatidylinositol (3)P levels. Analysis of MTM expression patterns using transcriptional green fluorescence protein reporters demonstrated that these two MTMs exhibit mostly non-overlapping expression patterns and that MTM-green fluorescence protein fusion proteins are localized to different subcellular locations. These observations suggest that some of the different functions of MTMs might, in part, be a consequence of unique expression and localization patterns. However, our finding that at least three C. elegans MTMs play essential roles in coelomocyte endocytosis, a process that also requires VPS34, indicates that MTMs do not simply turn off VPS34 but unexpectedly also function as positive regulators of biological processes.

KCa3.1 potassium channels are critical for cAMP-dependent chloride secretion and cyst growth in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by numerous fluid-filled kidney cysts. Net fluid secretion into renal cysts is caused by transepithelial transport mediated by the apical cystic fibrosis transmembrane conductance regulator chloride channel, which leads to cyst enlargement. Here we found that forskolin, a potent adenylyl cyclase agonist, stimulated anion secretion by monolayers of kidney cells derived from patients with ADPKD. TRAM-34, a specific KCa3.1 potassium channel blocker, inhibited this current, and in vitro cyst formation and enlargement by the cells cultured within a collagen gel. Net chloride secretion was enhanced by the KCa3.1 activator DCEBIO and both chloride secretion and in vitro cyst growth were inhibited by overexpression of myotubularin-related protein-6, a phosphatase that specifically inhibits KCa3.1 channel activity. Our study suggests that KCa3.1 channels play a critical role in transcellular chloride secretion and net fluid transport into the kidney cysts of patients with ADPKD by maintaining the electrochemical driving force for chloride efflux through apical chloride channels. Pharmacological inhibitors of KCa3.1 channels may provide a novel and effective therapy to delay progression to kidney failure in patients with ADPKD.

Nucleoside diphosphate kinase B knock-out mice have impaired activation of the K+ channel KCa3.1, resulting in defective T cell activation.

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K+ channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca2+ flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B−/− mice. NDPK-B−/− mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B−/− mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1−/− mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B−/− mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B−/− mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases.

The class II phosphatidylinositol 3 kinase C2β is required for the activation of the K+ channel KCa3.1 and CD4 T-cells

The Ca(2+)-activated K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of T-cells. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, directly phosphorylates and activates KCa3.1 and is required for the activation of human CD4 T lymphocytes. We now show that the class II phosphatidylinositol 3 kinase C2beta (PI3K-C2beta) is activated by the T-cell receptor (TCR) and functions upstream of NDPK-B to activate KCa3.1 channel activity. Decreased expression of PI3K-C2beta by siRNA in human CD4 T-cells resulted in inhibition of KCa3.1 channel activity. The inhibition was due to decreased phosphatidylinositol 3-phosphate [PI(3)P] because dialyzing PI3K-C2beta siRNA-treated T-cells with PI(3)P rescued KCa3.1 channel activity. Moreover, overexpression of PI3K-C2beta in KCa3.1-transfected Jurkat T-cells led to increased TCR-stimulated activation of KCa3.1 and Ca(2+) influx, whereas silencing of PI3K-C2beta inhibited both responses. Using total internal reflection fluorescence microscopy and planar lipid bilayers, we found that PI3K-C2beta colocalized with Zap70 and the TCR in peripheral microclusters in the immunological synapse. This is the first demonstration that a class II PI3K plays a critical role in T-cell activation.

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.