A TNFR1-UBCH10 axis drives lung squamous cell carcinoma dedifferentiation and metastasis through a cell-autonomous signaling loop.

Tumor necrosis factor receptor 1 (TNFR1), encoded by TNFRSF1A, is a critical transducer of inflammatory pathways, but its physiological role in human cancer is not completely understood. Here, we observed high expression of TNFR1 in many human lung squamous cell carcinoma (SCCs) samples and in spontaneous lung SCCs derived from kinase-dead Ikkα knock-in (KA/KA) mice. Knocking out Tnfrf1a in KA/KA mice blocked lung SCC formation. When injected via tail vein, KALLU+ lung SCC cells that highly expressed TNFR1/TNF, Sox2, c-Myc, Twist1, Bcl2, and UBCH10, generated dedifferentiated spindle cell carcinomas with epithelial-mesenchymal transition markers in mouse lungs. In contrast, KALLU+ cells with silenced TNFR1 and KALLU- cells that expressed low levels of TNFR1 generated well-differentiated lung SCCs and were less tumorigenic and metastatic. We identified a downstream effector of TNFR1: oncogenic UBCH10, an E2 ubiquitin-conjugating enzyme with targets including Twist1, c-Myc, and Sox2, which enhanced SCC cell dedifferentiation. Furthermore, Tg-K5.TNFR1;KA/KA mice, which expressed transgenic TNFR1 in keratin 5-positve epithelial cells, developed more poorly differentiated and metastatic lung SCCs than those found in KA/KA mice. These findings demonstrate that an overexpressed TNFR1-UBCH10 axis advances lung carcinogenesis and metastasis through a dedifferentiation mechanism. Constituents in this pathway may contribute to the development of differentiation-related therapies for lung SCC.

IKKα-deficient lung adenocarcinomas generate an immunosuppressive microenvironment by overproducing Treg-inducing cytokines.

The tumor microenvironment (TME) provides potential targets for cancer therapy. However, how signals originating in cancer cells affect tumor-directed immunity is largely unknown. Deletions in the CHUK locus, coding for IκB kinase α (IKKα), correlate with reduced lung adenocarcinoma (ADC) patient survival and promote KrasG12D-initiated ADC development in mice, but it is unknown how reduced IKKα expression affects the TME. Here, we report that low IKKα expression in human and mouse lung ADC cells correlates with increased monocyte-derived macrophage and regulatory T cell (Treg) scores and elevated transcription of genes coding for macrophage-recruiting and Treg-inducing cytokines (CSF1, CCL22, TNF, and IL-23A). By stimulating recruitment of monocyte-derived macrophages from the bone marrow and enforcing a TNF/TNFR2/c-Rel signaling cascade that stimulates Treg generation, these cytokines promote lung ADC progression. Depletion of TNFR2, c-Rel, or TNF in CD4+ T cells or monocyte-derived macrophages dampens Treg generation and lung tumorigenesis. Treg depletion also attenuates carcinogenesis. In conclusion, reduced cancer cell IKKα activity enhances formation of a protumorigenic TME through a pathway whose constituents may serve as therapeutic targets for KRAS-initiated lung ADC.

NLRP3 Inhibition Ameliorates Severe Cutaneous Autoimmune Manifestations in a Mouse Model of Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy-Like Disease.

Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy show diverse endocrine and nonendocrine manifestations initiated by self-reactive T cells because of AIRE mutation-induced defective central tolerance. A large number of American patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy suffer from early-onset cutaneous inflammatory lesions accompanied by an infiltration of T cells and myeloid cells. The role of myeloid cells in this setting remains to be fully investigated. In this study, we characterize the autoinflammatory phenotypes in the skin of both autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy-like kinase-dead Ikkα knockin mice and patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. We found a marked infiltration of autoreactive CD4 T cells, macrophages, and neutrophils; elevated uric acid; and increased NLRP3, a major inflammasome component. Depleting autoreactive CD4 T cells or ablating Ccl2/Cxcr2 genes significantly attenuated the inflammasome activity, inflammation, and skin phenotypes in kinase-dead Ikkα knockin mice. Importantly, treatment with an NLRP3 inhibitor reduced skin phenotypes and decreased infiltration of CD4 T cells, macrophages, and neutrophils. These results suggest that increased myeloid cell infiltration contributes to autoreactive CD4 T cell-mediated skin autoinflammation. Thus, our findings reveal that the combined infiltration of macrophages and neutrophils is required for autoreactive CD4 T cell-mediated skin disease pathogenesis and that the NLRP3-dependent inflammasome is a potential therapeutic target for the cutaneous manifestations of autoimmune diseases.

C-CBL is required for inhibition of angiogenesis through modulating JAK2/STAT3 activity in ROP development.

PURPOSE: The incidence of retinopathy of prematurity (ROP) has increased continuously in recent years. However, the therapeutic effects of current treatments still remain undesired. This study aims to investigate the role of C-CBL in retinal angiogenesis in ROP and its potential as a therapeutic target. METHODS: Mouse retina microvascular endothelial cells (mRMECs) and induced experimental ROP/ oxygen-induced retinopathy (OIR) mice were employed to investigate the role of C-CBL in angiogenesis with combined molecular and cellular approaches, and histopathology methods. OIR mouse pups at postnatal day 12 (P12) were either injected intravitreally with adenovirus overexpressing c-Cbl or c-Cbl siRNA. Retinal neovascularization and avascular status were evaluated by retinal immunofluorescence (IF) staining, whole-mounts and hematoxylin and eosin (H&E) staining. RESULTS: C-CBL inhibits neovascularization by negatively regulating JAK2/STAT3/VEGF signaling axis in a ubiquitination-dependent manner. Knockdown of c-Cbl by siRNA reduced ubiquitin-mediated JAK2 degradation and increased levels of p-JAK2, p-STAT3, VEGF, and neovascularization in mRMECs, which can be reversed by JAK2 inhibitor treatment. While knockdown of c-Cbl significantly increased neovascular (NV) zone in the retinas, c-Cbl overexpression inhibited neovascularization in the retinal tissues in OIR mice. CONCLUSION: We found that C-CBL is required for anti-neovascularization process in ROP development by inhibiting JAK2/STAT3-dependent angiogenesis. Thus, our finding strongly suggest that C-CBL may be a potential novel therapeutic target for treating ROP.

IKKα deficiency disrupts the development of marginal zone and follicular B cells.

Only few genes have been confidently identified to be involved in the Follicular (FO) and Marginal Zone (MZ) B cell differentiation, migration, and retention in the periphery. Our group previously observed that IKKα kinase inactive mutant mice IKKαK44A/K44A have significantly lower number of MZ B cells whereas FO B cell numbers appeared relatively normal. Because kinase dead IKKα can retain some of its biological functions that may interfere in revealing its actual role in the MZ and FO B cell differentiation. Therefore, in the current study, we genetically deleted IKKα from the pro-B cell lineage that revealed novel functions of IKKα in the MZ and FO B lymphocyte development. The loss of IKKα produces a significant decline in the percentage of immature B lymphocytes, mature marginal zone B cells, and follicular B cells along with a severe disruption of splenic architecture of marginal and follicular zones. IKKα deficiency affect the recirculation of mature B cells through bone marrow. A transplant of IKKα knockout fetal liver cells into Rag-/- mice shows a significant reduction compared to control in the B cells recirculating through bone marrow. To reveal the genes important in the B cell migration, a high throughput gene expression analysis was performed on the IKKα deficient recirculating mature B cells (B220+IgMhi). That revealed significant changes in the expression of genes involved in the B lymphocyte survival, homing and migration. And several among those genes identified belong to G protein family. Taken together, this study demonstrates that IKKα forms a vial axis controlling the genes involved in MZ and FO B cell differentiation and migration.

Macrophage inducible nitric oxide synthase circulates inflammation and promotes lung carcinogenesis.

Human lung squamous cell carcinoma (SCC) is highly associated with increased pulmonary macrophage infiltration. Previously, we showed that marked pulmonary infiltrating macrophages were required for spontaneous lung SCC development in a mouse model (L-IkkαKA/KA , KA/KA) that resembles human lung SCC. Interestingly the lung SCC-associated macrophages specifically express elevated inducible nitric oxide synthase (NOS2). However, the role of macrophage NOS2 in lung carcinogenesis has not been explored. Here, we show that NOS2 ablation inhibits macrophage infiltration, fibrosis, and SCC development in the lungs of KA/KA mice. Macrophage NOS2 was found to circulate inflammation and enhance macrophage migration and survival. NOS2 promotes foamy macrophage formation characterized with impaired lipid metabolism. NOS2 null bone marrow transplantation reduces foamy macrophage numbers and carcinogenesis in KA/KA chimaeras. This finding sheds light on a new mechanism by which macrophage NOS2 increases pulmonary inflammatory responses and macrophage survival and impairs macrophage lipid metabolism, thereby promoting lung SCC formation.

IKKα inactivation promotes Kras-initiated lung adenocarcinoma development through disrupting major redox regulatory pathways.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two distinct and predominant types of human lung cancer. IκB kinase α (IKKα) has been shown to suppress lung SCC development, but its role in ADC is unknown. We found inactivating mutations and homologous or hemizygous deletions in the CHUK locus, which encodes IKKα, in human lung ADCs. The CHUK deletions significantly reduced the survival time of patients with lung ADCs harboring KRAS mutations. In mice, lung-specific Ikkα ablation (IkkαΔLu ) induces spontaneous ADCs and promotes KrasG12D-initiated ADC development, accompanied by increased cell proliferation, decreased cell senescence, and reactive oxygen species (ROS) accumulation. IKKα deletion up-regulates NOX2 and down-regulates NRF2, leading to ROS accumulation and blockade of cell senescence induction, which together accelerate ADC development. Pharmacologic inhibition of NADPH oxidase or ROS impairs KrasG12D-mediated ADC development in IkkαΔLu mice. Therefore, IKKα modulates lung ADC development by controlling redox regulatory pathways. This study demonstrates that IKKα functions as a suppressor of lung ADC in human and mice through a unique mechanism that regulates tumor cell-associated ROS metabolism.

Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis.

Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell-driven autoimmune disease caused by impaired central tolerance, are susceptible to chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop APECED-like phenotypes, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or autoreactive CD4 T cell depletion rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or epidermal growth factor receptor (EGFR) activity decreases fungal burden. Fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development.

Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma.

Lung cancer is the leading cause of cancer death. Beyond first line treatment, few therapeutic options are available, particularly for squamous cell carcinoma (SCC). Here, we have explored the phospholipidomes of 30 human SCCs and found that they almost invariably (in 96.7% of cases) contain phospholipids with longer acyl chains compared to matched normal tissues. This trait was confirmed using in situ 2D-imaging MS on tissue sections and by phospholipidomics of tumor and normal lung tissue of the L-IkkαKA/KA mouse model of lung SCC. In both human and mouse, the increase in acyl chain length in cancer tissue was accompanied by significant changes in the expression of acyl chain elongases (ELOVLs). Functional screening of differentially expressed ELOVLs by selective gene knockdown in SCC cell lines followed by phospholipidomics revealed ELOVL6 as the main elongation enzyme responsible for acyl chain elongation in cancer cells. Interestingly, inhibition of ELOVL6 drastically reduced colony formation of multiple SCC cell lines in vitro and significantly attenuated their growth as xenografts in vivo in mouse models. These findings identify acyl chain elongation as one of the most common traits of lung SCC discovered so far and pinpoint ELOVL6 as a novel potential target for cancer intervention.

IKKα is required for the homeostasis of regulatory T cells and for the expansion of both regulatory and effector CD4 T cells.

It was reported that TNF receptor type II signaling, which has the capacity to stimulate CD4+ forkhead box P3+ (Foxp3+) regulatory T cells (Tregs), activated the noncanonical NF-κB pathway in an IKKα-dependent manner. Therefore, we studied the role of IKKα in the homeostasis of Treg population. To this end, we generated a mouse strain with conditional knockout of IKKα in CD4 cells (Ikkα(f/f):CD4.Cre) that showed a >60% reduction in the number of Tregs in the thymus and peripheral lymphoid tissues, whereas the number of Foxp3- effector T cells (Teffs) remained at a normal level. The function of Tregs deficient in IKKα was examined using Rag1(-/-) mice cotransferred with naive CD4 cells (nCD4s). Although wild-type (WT) Tregs inhibited colitis induced by transfer of WT nCD4s, IKKα-deficient Tregs failed to do so, which was associated with their inability to reconstitute Rag1(-/-) mice. Furthermore, nCD4s deficient in IKKα also failed to reconstitute Rag1(-/-) mice and were defective in proliferative responses in vitro and in vivo. Thus, our study reveals a novel role of IKKα in the maintenance of a normal Treg population and in the control of expansion of CD4 T cells. These properties of IKKα may be exploited as therapeutic strategies in the treatment of major human diseases.

IKKα negatively regulates ASC-dependent inflammasome activation.

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IκB kinase α (IKKα) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKα controls the inflammasome at the level of the adaptor ASC, which interacts with IKKα in the nucleus of resting macrophages in an IKKα kinase-dependent manner. Loss of IKKα kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKα in the perinuclear area following translocation of the ASC/IKKα complex. Signal 2 of NLRP3 activation leads to inhibition of IKKα kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKα-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

An IKKα-nucleophosmin axis utilizes inflammatory signaling to promote genome integrity.

The inflammatory microenvironment promotes skin tumorigenesis. However, the mechanisms by which cells protect themselves from inflammatory signals are unknown. Downregulation of IKKα promotes skin tumor progression from papillomas to squamous cell carcinomas, which is frequently accompanied by genomic instability, including aneuploid chromosomes and extra centrosomes. In this study, we found that IKKα promoted oligomerization of nucleophosmin (NPM), a negative centrosome duplication regulator, which further enhanced NPM and centrosome association, inhibited centrosome amplification, and maintained genome integrity. Levels of NPM hexamers and IKKα were conversely associated with skin tumor progression. Importantly, proinflammatory cytokine-induced IKKα activation promoted the formation of NPM oligomers and reduced centrosome numbers in mouse and human cells, whereas kinase-dead IKKα blocked this connection. Therefore, our findings suggest a mechanism in which an IKKα-NPM axis may use inflammatory signals to suppress centrosome amplification, promote genomic integrity, and prevent tumor progression.

The pivotal role of IKKα in the development of spontaneous lung squamous cell carcinomas.

Here, we report that kinase-dead IKKα knockin mice develop spontaneous lung squamous cell carcinomas (SCCs) associated with IKKα downregulation and marked pulmonary inflammation. IKKα reduction upregulated the expression of p63, Trim29, and keratin 5 (K5), which serve as diagnostic markers for human lung SCCs. IKKα(low)K5(+)p63(hi) cell expansion and SCC formation were accompanied by inflammation-associated deregulation of oncogenes, tumor suppressors, and stem cell regulators. Reintroducing transgenic K5.IKKα, depleting macrophages, and reconstituting irradiated mutant animals with wild-type bone marrow (BM) prevented SCC development, suggesting that BM-derived IKKα mutant macrophages promote the transition of IKKα(low)K5(+)p63(hi) cells to tumor cells. This mouse model resembles human lung SCCs, sheds light on the mechanisms underlying lung malignancy development, and identifies targets for therapy of lung SCCs.

IKKα-mediated signaling circuitry regulates early B lymphopoiesis during hematopoiesis.

Multiple transcription factors regulate B-cell commitment, which is coordinated with myeloid-erythroid lineage differentiation. NF-κB has long been speculated to regulate early B-cell development; however, this issue remains controversial. IκB kinase-α (IKKα) is required for splenic B-cell maturation but not for BM B-cell development. In the present study, we unexpectedly found defective BM B-cell development and increased myeloid-erythroid lineages in kinase-dead IKKα (KA/KA) knock-in mice. Markedly increased cytosolic p100, an NF-κB2-inhibitory form, and reduced nuclear NF-κB p65, RelB, p50, and p52, and IKKα were observed in KA/KA splenic and BM B cells. Several B- and myeloid-erythroid-cell regulators, including Pax5, were deregulated in KA/KA BM B cells. Using fetal liver and BM congenic transplantations and deleting IKKα from early hematopoietic cells in mice, this defect was identified as being B cell-intrinsic and an early event during hematopoiesis. Reintroducing IKKα, Pax5, or combined NF-κB molecules promoted B-cell development but repressed myeloid-erythroid cell differentiation in KA/KA BM B cells. The results of the present study demonstrate that IKKα regulates B-lineage commitment via combined canonical and noncanonical NF-κB transcriptional activities to target Pax5 expression during hematopoiesis.

Reduction of IKKalpha expression promotes chronic ultraviolet B exposure-induced skin inflammation and carcinogenesis.

Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor kappaB kinase-alpha (IKKalpha) plays an important role in maintaining skin homeostasis, and expression of IKKalpha was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKalpha in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkalpha(+/+) and Ikkalpha(+/-) mice. Ikkalpha(+/-) mice were found to develop a twofold greater number of skin tumors than Ikkalpha(+/+) mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkalpha(+/-) than in Ikkalpha(+/+) mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkalpha(+/-) compared with those in Ikkalpha(+/+) skin. Also, reduction of IKKalpha levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFalpha, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKalpha expression orchestrates UVB carcinogen, accelerating tumorigenesis.

The influence of IL-2 family cytokines on activation and function of naturally occurring regulatory T cells.

IL-2 is essential for CD4+CD25+forkhead box P3+ (FoxP3+) naturally occurring regulatory T cell (Treg) homeostasis and activation. Binding of IL-2 to its receptor leads to phosphorylation of STAT5, and binding of phosphorylated STAT5 to the foxp3 promoter increases foxp3 transcription, resulting in elevated levels of FoxP3 protein in Tregs. Transcriptional regulation by the elevated levels of FoxP3 is thought to be essential for the strong suppressor function seen in activated Tregs. IL-2 belongs to a family cytokines, which all depend on the common gamma-receptor chain (gammac). Given the well-documented effects of IL-2 on Treg function, the effect of other IL-2 family cytokines (IL-7, -15, and -21) on Tregs was examined. We observed that IL-7 and IL-15 induce STAT5 phosphorylation and up-regulation of FoxP3 in Tregs. STAT5 activation correlated with enhanced viability. However, only in the presence of IL-2 did Tregs acquire potent suppressor function. This finding is surprising, as IL-15 as well as IL-2 use the same IL-2R betac and gammac for signaling. In contrast, IL-21 activated STAT3 but did not activate STAT5 and had no effect on Treg viability, activation, or function. We therefore conclude that phosphorylation of STAT5, mediated through the IL-2Rgamma, promotes Treg survival in a resting and activated state. However, activation of STAT5 alone in conjunction with TCR signaling is not sufficient for the induction of potent suppressor function in Tregs, as IL-7 and IL-15 are not capable of inducing potent Treg suppressor function.

Development of regulatory T cells requires IL-7Ralpha stimulation by IL-7 or TSLP.

Interleukin-7 (IL-7), a cytokine produced by stromal cells, is required for thymic development and peripheral homeostasis of most major subsets of T cells. We examined whether regulatory T (Treg) cells also required the IL-7 pathway by analyzing IL-7Ralpha(-/-) mice. We observed a striking reduction in cells with the Treg surface phenotype (CD4, CD25, GITR (glucocorticoid-induced tumor necrosis factor [TNF]-like receptor), CD45RB, CD62L, CD103) or intracellular markers (cytotoxic T-lymphocyte-associated antigen-4, CTLA-4, and forkhead box transcription factor 3, Foxp3). Foxp3 transcripts were virtually absent in IL-7Ralpha(-/-) lymphoid tissues, and no Treg cell suppressive activity could be detected. There are 2 known ligands for IL-7Ralpha: IL-7 itself and thymic stromal lymphopoietin (TSLP). Surprisingly, mice deficient in IL-7 or the other chain of the TSLP receptor (TSLPR) developed relatively normal numbers of Treg cells. Combined deletion of IL-7 and TSLP receptor greatly reduced Treg cell development in the thymus but was not required for survival of mature peripheral Treg cells. We conclude that Treg cells, like other T cells, require signals from the IL-7 receptor, but unlike other T cells, do not require IL-7 itself because of at least partially overlapping actions of IL-7 and TSLP for development of Treg cells.

Modulation of lymphocyte function with inhibitory CD2: loss of NK and NKT cells.

Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8+ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.

Regulation of Ly49D/DAP12 signal transduction by Src-family kinases and CD45.

Activating, DAP12-coupled members of the Ly-49 family of NK cell receptors help control viral infections in mice. However, the kinases and/or phosphatases mediating tyrosine phosphorylation of Ly-49D-associated DAP12 have not been elucidated. In this study, we show for the first time that Src family tyrosine kinases are physically and functionally associated with Ly-49D/DAP12 signaling in murine NK cells. Specifically, we demonstrate the following: 1) inhibition of Src family kinases suppresses DAP12 phosphorylation and downstream DAP12 signals; 2) both Fyn and Lck are capable of phosphorylating DAP12; and 3) both kinases coimmunoprecipitate with the Ly-49D/DAP12 complex in NK cells. Although we detect enhanced phosphorylation of Fyn upon Ly-49D cross-linking in NK cells, Ly-49D-mediated events in both Fyn-/- and Fyn/Lck-/- mice appear normal, reinforcing the theme of redundancy in the ability of Src family kinases to initiate activation events. In contrast to disruption of specific Src family enzymes, Ly-49D/DAP12-mediated calcium mobilization and cytokine production by CD45 null NK cells are defective. Although others have ascribed the effects of CD45 mutation solely on the suppression of Src family activity, we demonstrate in this study that DAP12 is hyperphosphorylated in CD45 null NK cells, resulting in uncoordinated tyrosine-mediated signaling upon Ly-49D ligation. Therefore, although our data are consistent with a Src kinase activity proximally within DAP12 signaling, DAP12 also appears to be a substrate of CD45, suggesting a more complex role for this phosphatase than has been reported previously.