Mast cells promote small bowel cancer in a tumor stage-specific and cytokine-dependent manner.

Mast cells (MCs) are tissue resident sentinels that mature and orchestrate inflammation in response to infection and allergy. While they are also frequently observed in tumors, the contribution of MCs to carcinogenesis remains unclear. Here, we show that sequential oncogenic events in gut epithelia expand different types of MCs in a temporal-, spatial-, and cytokine-dependent manner. The first wave of MCs expands focally in benign adenomatous polyps, which have elevated levels of IL-10, IL-13, and IL-33, and are rich in type-2 innate lymphoid cells (ILC2s). These vanguard MCs adhere to the transformed epithelial cells and express murine mast cell protease 2 (mMCP2; a typical mucosal MC protease) and, to a lesser extent, the connective tissue mast cell (CTMC) protease mMCP6. Persistence of MCs is strictly dependent on T cell-derived IL-10, and their loss in the absence of IL-10-expressing T cells markedly delays small bowel (SB) polyposis. MCs expand profusely in polyposis-prone mice when T cells overexpress IL-10. The frequency of polyp-associated MCs is unaltered in response to broad-spectrum antibiotics, arguing against a microbial component driving their recruitment. Intriguingly, when polyps become invasive, a second wave of mMCP5+/mMCP6+ CTMCs expands in the tumor stroma and at invasive tumor borders. Ablation of mMCP6 expression attenuates polyposis, but invasive properties of the remaining lesions remain intact. Our findings argue for a multistep process in SB carcinogenesis in which distinct MC subsets, and their elaborated proteases, guide disease progression.

Preferential expansion of pro-inflammatory Tregs in human non-small cell lung cancer.

OBJECTIVES: Lung cancer is the leading cause of cancer-related death in the USA. Regulatory T cells (Tregs) normally function to temper immune responses and decrease inflammation. Previous research has demonstrated different subsets of Tregs with contrasting anti- or pro-inflammatory properties. This study aimed to determine Treg subset distributions and characteristics present in non-small cell lung cancer (NSCLC) patients. METHODS: Peripheral blood was collected from healthy controls (HC) and NSCLC patients preceding surgical resection, and mononuclear cells were isolated, stained, and analyzed by flow cytometry. Tregs were defined by expression of CD4 and CD25 and classified into CD45RA(+)Foxp3(int) (naïve, Fr. I) or CD45RA(-)Foxp3(hi) (activated Fr. II). Activated conventional T cells were CD4(+)CD45RA(-)Foxp3(int) (Fr. III). RESULTS: Samples from 23 HC and 26 NSCLC patients were collected. Tregs isolated from patients with NSCLC were found to have enhanced suppressive function on naive T cells. Cancer patients had significantly increased frequencies of activated Tregs (fraction II: FrII), 17.5 versus 3.2% (P < 0.001). FrII Tregs demonstrated increased RORγt and IL17 expression and decreased IL10 expression compared to Tregs from HC, indicating pro-inflammatory characteristics. CONCLUSIONS: This study demonstrates that a novel subset of Tregs with pro-inflammatory characteristics preferentially expand in NSCLC patients. This Treg subset appears identical to previously reported pro-inflammatory Tregs in human colon cancer patients and in mouse models of polyposis. We expect the pro-inflammatory Tregs in lung cancer to contribute to the immune pathogenesis of disease and propose that targeting this Treg subset may have protective benefits in NSCLC.

T-cell Expression of IL10 Is Essential for Tumor Immune Surveillance in the Small Intestine.

IL10 is attributed with immune-suppressive and anti-inflammatory properties, which could promote or suppress cancer in the gastrointestinal tract. Loss of IL10 exacerbates colonic inflammation, leading to colitis and cancer. Consistent with this, transfer of IL10-competent regulatory T cells (Treg) into mice with colitis or hereditary polyposis protects against disease, while IL10-deficient mice are predisposed to polyposis with increased colon polyp load. Little is known about the protective or pathogenic function of IL10 in cancers of the small intestine. We found CD4(+) T cells and CD4(+) Foxp3(+) Tregs to be the major sources of IL10 in the small intestine and responsible for the increase in IL10 during polyposis in the APC(Δ468) mouse model of hereditary polyposis. Targeted ablation of IL10 in T cells caused severe IL10 deficiency and delayed polyp growth. However, these polyps progressively lost cytotoxic activity and eventually progressed to cancer. Several observations suggested that the effect was due to the loss of IFNγ-dependent immune surveillance. IL10-incompetent CD4(+) T cells failed to secrete IFNγ when stimulated with polyp antigens and were inefficient in T-helper-1 (TH1) commitment. By contrast, the TH17 commitment was unaffected. These findings were validated using mice whose T cells overexpress IL10. In these mice, we observed high intra-polyp cytotoxic activity and attenuation of polyposis. Thus, expression of IL10 by T cells is protective and required for immune surveillance in the small intestine.

Zileuton, 5-lipoxygenase inhibitor, acts as a chemopreventive agent in intestinal polyposis, by modulating polyp and systemic inflammation.

PURPOSE: Leukotrienes and prostaglandins, products of arachidonic acid metabolism, sustain both systemic and lesion-localized inflammation. Tumor-associated Inflammation can also contribute to the pathogenesis of colon cancer. Patients with inflammatory bowel disease (IBD) have increased risk of developing colon cancer. The levels of 5-lipoxygenase (5-LO), the key enzyme for leukotrienes production, are increased in colon cancer specimens and colonic dysplastic lesions. Here we report that Zileuton, a specific 5-LO inhibitor, can prevent polyp formation by efficiently reducing the tumor-associated and systemic inflammation in APCΔ468 mice. EXPERIMENTAL DESIGN: In the current study, we inhibited 5-LO by dietary administration of Zileuton in the APCΔ468 mouse model of polyposis and analyzed the effect of in vivo 5-LO inhibition on tumor-associated and systemic inflammation. RESULTS: Zileuton-fed mice developed fewer polyps and displayed marked reduction in systemic and polyp-associated inflammation. Pro-inflammatory cytokines and pro-inflammatory innate and adaptive immunity cells were reduced both in the lesions and systemically. As part of tumor-associated inflammation Leukotriene B4 (LTB4), product of 5-LO activity, is increased focally in human dysplastic lesions. The 5-LO enzymatic activity was reduced in the serum of Zileuton treated polyposis mice. CONCLUSIONS: This study demonstrates that dietary administration of 5-LO specific inhibitor in the polyposis mouse model decreases polyp burden, and suggests that Zileuton may be a potential chemo-preventive agent in patients that are high-risk of developing colon cancer.

Methods for the study of mast cells in cancer.

Tumor growth requires interactions of tumor cells with a receptive and inductive microenvironment. Two major populations of tumor-infiltrating cells are considered to be essential for producing such a microenvironment: (1) proinflammatory cells that nurture the tumor with growth factors and facilitate invasion and metastasis by secreting proteases and (2) immune suppressive leukocytes including T-regulatory cells (Treg) that hinder tumor-specific CD8 T-cell responses, which otherwise could potentially reject the tumor. Among the proinflammatory cells, accumulation of mast cells (MCs) in human tumors is frequently recorded and was recently linked with poor prognosis. Causative links between mast cell infiltration and tumor progression can be deduced from animal studies. There is an interesting link between mast cells and Treg. The adoptive transfer of Treg from healthy syngeneic mice to mice susceptible to colon cancer suppresses focal mastocytosis and hinders tumor progression. Furthermore, T-cell-deficient mice susceptible to colon cancer show enhanced focal mastocytosis and tumor invasion. Here, we describe methods to assess MCs in mouse models of cancer and to investigate how MCs affect tumor epithelium. Additionally, we will detail methods used to investigate how T cells influence MCs and how MCs influence T cells.

Current status of interleukin-10 and regulatory T-cells in cancer.

PURPOSE OF REVIEW: Tumor growth elicits antigen-specific cytotoxic as well as immune suppressive responses. Interleukin-10 (IL-10) is a key immune-suppressive cytokine produced by regulatory T-cells and by helper T-cells. Here, we review pleiotropic functions of IL-10 that impact the immune pathology of cancer. RECENT FINDINGS: The role of IL-10 in cancer has become less certain with the knowledge of its immune stimulatory functions. IL-10 is needed for T-helper cell functions, T-cell immune surveillance, and suppression of cancer-associated inflammation. By promoting tumor-specific immune surveillance and hindering pathogenic inflammation, IL-10 is emerging as a key cytokine in the battle of the host against cancer. SUMMARY: IL-10 functions at the cross-roads of immune stimulation and immune suppression in cancer. Immunological mechanisms of action of IL-10 can be ultimately exploited to develop novel and effective cancer therapies.

Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10-producing T cells.

Interleukin (IL)-10 is elevated in cancer and is thought to contribute to immune tolerance and tumor growth. Defying these expectations, the adoptive transfer of IL-10-expressing T cells to mice with polyposis attenuates microbial-induced inflammation and suppresses polyposis. To gain better insights into how IL-10 impacts polyposis, we genetically ablated IL-10 in T cells in APC(Δ468) mice and compared the effects of treatment with broad-spectrum antibiotics. We found that T cells and regulatory T cells (Treg) were a major cellular source of IL-10 in both the healthy and polyp-bearing colon. Notably, T cell-specific ablation of IL-10 produced pathologies that were identical to mice with a systemic deficiency in IL-10, in both cases increasing the numbers and growth of colon polyps. Eosinophils were found to densely infiltrate colon polyps, which were enriched similarly for microbiota associated previously with colon cancer. In mice receiving broad-spectrum antibiotics, we observed reductions in microbiota, inflammation, and polyposis. Together, our findings establish that colon polyposis is driven by high densities of microbes that accumulate within polyps and trigger local inflammatory responses. Inflammation, local microbe densities, and polyp growth are suppressed by IL-10 derived specifically from T cells and Tregs.

The two faces of regulatory T cells in cancer.

Regulatory T cells (Tregs) that expand in human colon cancer express retinoid-related orphan receptor γt (RORγt) and exert potent T-cell suppressive functions while mediating pro-inflammatory effects. Similar Tregs expand and drive a vicious cycle of inflammation in murine polyposis. Targeting RORγt in Tregs interrupts such a cycle and protects mice against polyposis, suggesting that a similar intervention may provide therapeutic benefits to colon cancer patients.

PI3K/AKT signaling is essential for communication between tissue-infiltrating mast cells, macrophages, and epithelial cells in colitis-induced cancer.

PURPOSE: To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy for chronic inflammatory diseases. We have explored phosphoinositide 3-kinase (PI3K) activity in human inflammatory bowel diseases and mouse colitis models. EXPERIMENTAL DESIGN: We conducted immunostaining of phosphorylated AKT (pAKT) and unbiased high-throughput image acquisition and quantitative analysis of samples of noninflamed normal colon, colitis, dysplasia, and colorectal cancer. Mechanistic insights were gained from ex vivo studies of cell interactions, the piroxicam/IL-10(-/-) mouse model of progressive colitis, and use of the PI3K inhibitor LY294002. RESULTS: Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAM) and increase in densities of mast cells in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. Mast cells recruited macrophages in ex vivo migration assays, and both mast cells and TAMs promoted invasion of cancer cells. Pretreatment of mast cells with LY294002 blocked recruitment of TAMs. LY294002 inhibited mast cell and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer. CONCLUSION: The PI3K/AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feedback loop. The PI3K/AKT pathway is essential for tumor invasion and the malignant features of the piroxicam/IL-10(-/-) mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002.

Quantitative magnetic resonance imaging in the transgenic APC(Δ468) mouse model of hereditary colon cancer.

In this study, we investigated the use of high-resolution magnetic resonance imaging (MRI) methods for in vivo detection and quantitative characterization of colorectal tumors in the transgenic APC(Δ468) mouse model. High-resolution T(1)-weighted (T(1)W) images, T(2)-weighted (T(2)W) images, and dynamic contrast-enhanced (DCE) measurements were performed using a 7.0 T small-animal imaging system (N = 10). Individual tumors were identified on both T(1)W and T(2)W images. Twenty-eight tumors (2.8 ± 0.9 mm, mean ± SD) were detected with high-resolution MRI across a distance of roughly 3 cm from the rectum to the splenic flexure, whereas 29 tumors were found within corresponding colon tissue samples examined at gross necropsy in the same area. T(2) values were significantly different between tumor, skeletal muscle, and normal intestinal wall tissues (p < .05). For analysis of the vascular characteristics of colon tumor tissues using DCE measurements, the initial area under the curve (IAUC) for Gd contrast concentration curve (time) (C(Gd) [t]) was calculated with integration times of 60 and 120 seconds post-contrast infusion; two integration times were selected to capture both tracer wash-in and wash-out characteristics. IAUC measurements were significantly larger in tumor tissues compared to both normal intestinal wall and skeletal muscle tissues (p < .001). In vivo anatomic and quantitative MRI measurements were readily feasible in the transgenic APC(Δ468) mouse model. These noninvasive methods should improve experimental efficiencies during longitudinal survival studies that otherwise would require single-end-point necropsy measurements.

Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins.

Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.

Expression of RORγt marks a pathogenic regulatory T cell subset in human colon cancer.

The role of regulatory T cells (T(regs)) in human colon cancer (CC) remains controversial: high densities of tumor-infiltrating T(regs) can correlate with better or worse clinical outcomes depending on the study. In mouse models of cancer, T(regs) have been reported to suppress inflammation and protect the host, suppress T cells and protect the tumor, or even have direct cancer-promoting attributes. These different effects may result from the presence of different T(reg) subsets. We report the preferential expansion of a T(reg) subset in human CC with potent T cell-suppressive, but compromised anti-inflammatory, properties; these cells are distinguished from T(regs) present in healthy donors by their coexpression of Foxp3 and RORγt. T(regs) with similar attributes were found to be expanded in mouse models of hereditary polyposis. Indeed, ablation of the RORγt gene in Foxp3(+) cells in polyp-prone mice stabilized T(reg) anti-inflammatory functions, suppressed inflammation, improved polyp-specific immune surveillance, and severely attenuated polyposis. Ablation of interleukin-6 (IL-6), IL-23, IL-17, or tumor necrosis factor-α in polyp-prone mice reduced polyp number but not to the same extent as loss of RORγt. Surprisingly, loss of IL-17A had a dual effect: IL-17A-deficient mice had fewer polyps but continued to have RORγt(+) T(regs) and developed invasive cancer. Thus, we conclude that RORγt has a central role in determining the balance between protective and pathogenic T(regs) in CC and that T(reg) subtype regulates inflammation, potency of immune surveillance, and severity of disease outcome.

Abating colon cancer polyposis by Lactobacillus acidophilus deficient in lipoteichoic acid.

An imbalance of commensal bacteria and their gene products underlies mucosal and, in particular, gastrointestinal inflammation and a predisposition to cancer. Lactobacillus species have received considerable attention as examples of beneficial microbiota. We have reported previously that deletion of the phosphoglycerol transferase gene that is responsible for lipoteichoic acid (LTA) biosynthesis in Lactobacillus acidophilus (NCK2025) rendered this bacterium able to significantly protect mice against induced colitis when delivered orally. Here we report that oral treatment with LTA-deficient NCK2025 normalizes innate and adaptive pathogenic immune responses and causes regression of established colonic polyps. This study reveals the proinflammatory role of LTA and the ability of LTA-deficient L. acidophilus to regulate inflammation and protect against colonic polyposis in a unique mouse model.

The significant role of mast cells in cancer.

Mast cells (MC) are a bone marrow-derived, long-lived, heterogeneous cellular population that function both as positive and negative regulators of immune responses. They are arguably the most productive chemical factory in the body and influence other cells through both soluble mediators and cell-to-cell interaction. MC are commonly seen in various tumors and have been attributed alternatively with tumor rejection or tumor promotion. Tumor-infiltrating MC are derived both from sentinel and recruited progenitor cells. MC can directly influence tumor cell proliferation and invasion but also help tumors indirectly by organizing its microenvironment and modulating immune responses to tumor cells. Best known for orchestrating inflammation and angiogenesis, the role of MC in shaping adaptive immune responses has become a focus of recent investigations. MC mobilize T cells and antigen-presenting dendritic cells. They function as intermediaries in regulatory T cells (Treg)-induced tolerance but can also modify or reverse Treg-suppressive properties. The central role of MC in the control of innate and adaptive immunity endows them with the ability to tune the nature of host responses to cancer and ultimately influence the outcome of disease and fate of the cancer patient.

Mast cell 5-lipoxygenase activity promotes intestinal polyposis in APCDelta468 mice.

Arachidonic acid metabolism has been implicated in colon carcinogenesis, but the role of hematopoietic 5-lipoxygenase (5LO) that may impact tumor immunity in development of colon cancer has not been explored. Here we show that tissue-specific deletion of the 5LO gene in hematopoietic cells profoundly attenuates polyp development in the APC(Δ468) murine model of colon polyposis. In vitro analyses indicated that mast cells in particular utilized 5LO to limit proliferation of intestinal epithelial cells and to mobilize myeloid-derived suppressor cells (MDSCs). Mice lacking hemapoietic expression of 5LO exhibited reduced recruitment of MDSCs to the spleen, mesenteric lymph nodes, and primary tumor site. 5LO deficiency also reduced the activity in MDSCs of arginase-1, which is thought to be critical for MDSC function. Together, our results establish a pro-tumorigenic role of hematopoietic 5LO in the immune microenvironment and suggest 5LO inhibition as an avenue for future investigation in treatment of colorectal polyposis and cancer.

In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction.

T-regulatory cells (Treg) and mast cells (MC) are abundant in colorectal cancer (CRC) tumors. Interaction between the two is known to promote immune suppression or loss of Treg functions and autoimmunity. Here, we demonstrate that in both human CRC and murine polyposis the outcome of this interaction is the generation of potently immune suppressive but proinflammatory Treg (DeltaTreg). These Treg shut down IL10, gain potential to express IL17, and switch from suppressing to promoting MC expansion and degranulation. This change is also brought about by direct coculture of MC and Treg, or culture of Treg in medium containing IL6 and IL2. IL6 deficiency in the bone marrow of mice susceptible to polyposis eliminated IL17 production by the polyp infiltrating Treg, but did not significantly affect the growth of polyps or the generation of proinflammatory Treg. IL6-deficient MC could generate proinflammatory Treg. Thus, MC induce Treg to switch function and escalate inflammation in CRC without losing T-cell-suppressive properties. IL6 and IL17 are not needed in this process.

Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis.

BACKGROUND & AIMS: Mesalamine is a mainstay therapeutic agent in chronic ulcerative colitis (CUC) in which condition it reverses crypt architectural changes and reduces colitis-associated cancer (CAC). The present study addressed the possibility that mesalamine reduces beta-catenin-associated progenitor cell activation, Akt-phosphorylated beta-catenin(Ser552) (P-beta-catenin), and colitis-induced dysplasia (CID). METHODS: Effects of mesalamine on P-beta-catenin staining and function were assessed by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in biopsy specimens of CUC in mild or "refractory" severe mucosal inflammation. Effects of mesalamine on epithelial proliferation and activation of Akt and beta-catenin were assessed in interleukin (IL)-10(-/-) colitis and CID by immunohistochemistry and Western blotting. Dysplasia was assessed by counting the number and lengths of lesions per colon. RESULTS: Data from IL-10(-/-) and human colitis samples show that mesalamine reduced Akt activation and P-beta-catenin levels in the middle and upper crypt. Reductions in P-beta-catenin in CUC biopsy specimens with severe inflammation suggested that mesalamine reduced P-beta-catenin levels in tissue refractory to mesalamine's anti-inflammatory effects. In IL-10(-/-) mice, mesalamine reduced CID concordant with inhibition of crypt Akt and beta-catenin signaling. CONCLUSIONS: The results are consistent with the model that mesalamine contributes to chemoprevention in CAC by reducing beta-catenin signaling within intestinal progenitors.

T-regulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis.

T-regulatory (Treg) cells play a major role in cancer by suppressing protective antitumor immune responses. A series of observations (from a single laboratory) suggest that Treg cells are protective in cancer by virtue of their ability to control cancer-associated inflammation in an interleukin (IL)-10-dependent manner. Here, we report that the ability of Treg cells to produce IL-10 and control inflammation is lost in the course of progressive disease in a mouse model of hereditary colon cancer. Treg cells that expand in adenomatous polyps no longer produce IL-10 and instead switch to production of IL-17. Aberrant Treg cells from polyp-ridden mice promote rather than suppress focal mastocytosis, a critical tumor-promoting inflammatory response. The cells, however, maintain other Treg characteristics, including their inability to produce IL-2 and ability to suppress proliferation of stimulated CD4 T cells. By promoting inflammation and suppressing T-helper functions, these cells act as a double-edged knife propagating tumor growth.

The structural basis of novel endosome anchoring activity of KIF16B kinesin.

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Ctheta.

Protein kinase C (PKC) is a novel PKC that plays a key role in T lymphocyte activation. PKC has been shown to be specifically recruited to the immunological synapse in response to T cell receptor activation. To understand the basis of its unique subcellular localization properties, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKC. PKC showed phosphatidylserine selectivity in membrane binding and kinase action, which contributes to its translocation to the phosphatidylserine-rich plasma membrane in HEK293 cells. Unlike any other PKCs characterized so far, the isolated C1B domain of PKC had much higher affinity for DAG-containing membranes than the C1A domain. Also, the mutational analysis indicates that the C1B domain plays a predominant role in the DAG-induced membrane binding and activation of PKC. Furthermore, the Ca(2+)-independent C2 domain of PKC has significant affinity for anionic membranes, and the truncation of the C2 domain greatly enhanced the membrane affinity and enzyme activity of PKC. In addition, membrane binding properties of Y90E and Y90F mutants indicate that phosphorylation of Tyr(90) of the C2 domain enhances the affinity of PKC for model and cell membranes. Collectively, these results show that PKC has a unique membrane binding and activation mechanism that may account for its subcellular targeting properties.