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2؉-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways.A critical aspect of protein and/or lipid kinase regulation is the tight coupling of catalytic activity to various extracellular or intracellular signals. Alterations in levels of intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) 1 mediate the action of several hormones through the regulation of key enzymes; these signaling events are often orchestrated by the Ca 2ϩ effector, calmodulin (CaM) (1). Activation of phosphatidylinositol 3-kinase (PI3-kinase) in many tissues and cell types is required for mitogenesis, neuronal differentiation, and enhanced glucose transport (2). PI3-kinase and CaM are common components of several fundamental intracellular processes. For example, wortmannin, an inhibitor of PI3-kinase, and CGS9343B impede insulin-induced glucose uptake (3-5). Both CaM (6, 7) and PI3-kinase (8) participate in early endosome fusion. In the cytoskeleton, PI3-kinase has been linked to actin rearrangement (9), binds ␣-, ␤-and ␥-tubulin (10), and plays a role in platelet-derived growth factor-and insulin-induced membrane ruffling (11,12). Overexpression of CaM alters cell morphology and the arrangement of microfilaments within the cell (13). In addition, CaM binds to a variety of cytoskeletal proteins (1) including the family of unconventional myosins (14) and has been implicated in osteoclast membrane ruffling (15) and the formation of microspikes in neuronal cells (16). Finally, both PI3-kinase (17) and CaM, via modulation of the association of IQGAP1 with Cdc42 (18), may participate in the regulation of Rho family GTPases. Since CaM and PI3-kinase modulate similar cellular events, we evaluated a possible interaction between these two signaling components. EXPERIMENTAL PROCEDURESCell Culture and Lysis-Sf9 cells were maintained in Grace's medium supplemented with 10% fetal bovine serum and infected with baculovirus as described previously (19). CHO cells were grown to 80% confluence in Ham's F-12 medium with 10% fetal bovine serum. 32D cells and 32D cells expressing rat IRS-1 (32D/IRS-1) were cultured as described previously (20). The medium was removed, cells were washed 3 times with phosphate-buffered saline, and 1 ml of lysis buffer (50 mM Tris base, pH 7.4, 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 0.1 g/ml leupeptin, aprotonin, and pepstatin, and 1% Triton X-100) was added. Where indicated CaCl 2 or EGTA was included. The cells were collected and quick-frozen in methanol/dry CO 2 .Antibodies-The specific anti-calmodulin monoclonal antibody has been previously described (21). The anti-myoglobin monoclonal antibody (IgG 1 ) was highly purified by Dr. J. Ladenson (Washington University Medical Center, St. Louis). The anti-p85 antibody was prepared by immunizing rabbits with a glutathione S-transferase (GST) fusion protein containing the inter...

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Mixed lineage kinase 7 (MLK7) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the pro-apoptotic signaling pathways p38 and JNK. A library of potential kinase inhibitors was screened, and a series of dihydropyrrolopyrazole quinolines was identified as highly potent inhibitors of MLK7 in vitro catalytic activity. Of this series, an aryl-substituted dihydropyrrolopyrazole quinoline (DHP-2) demonstrated an IC 50 of 70 nM for inhibition of pJNK formation in COS-7 cell MLK7/JNK co-transfection assays. In stimulated cells, DHP-2 at 200 nM or MLK7 small interfering RNA completely blocked anisomycin and UV induced but had no effect on interleukin-1␤ or tumor necrosis factor-␣-induced p38 and JNK activation. Additionally, the compound blocked anisomycin and UV-induced apoptosis in COS-7 cells. Heart tissue homogenates from MLK7 transgenic mice treated with DHP-2 at 30 mg/kg had reduced JNK and p38 activation with no apparent effect on ERK activation, demonstrating that this compound can be used to block MLK7-driven MAPK pathway activation in vivo. Taken together, these data demonstrate that MLK7 is the MAPKKK required for modulation of the stressactivated MAPKs downstream of anisomycin and UV stimulation and that DHP-2 can be used to block MLK7 pathway activation in cells as well as in vivo.The mitogen-activated protein kinases (MAPK) 1 are a highly conserved family of signal transduction molecules that transmits extracellular signals from the membrane to the nucleus. There are three major branches of MAPK signaling that include ERK, c-Jun N-terminal kinase (JNK) and p38. The JNK and p38 branches of the MAPK family are activated by stress stimuli including cytokines, osmotic stress, mitogens, UV irradiation, chemotherapeutic agents, and anisomycin (1, 2). There are three kinases that form a MAPK signaling module where a MAPK is activated by a MAPK kinase (MAPKK), which in turn is regulated by a MAPKK kinase (MAPKKK) (Fig. 1). The upstream activation of JNK and p38 is complex, allowing for activation of this pathway in multiple cells and by multiple stimuli. Cellular and receptor specificity of the pathway is conferred by protein-protein interactions where the MAPK and a MAPKK assemble with a specific MAPKKK on scaffold proteins such as JNK-interacting protein (3, 4) or ␤-arrestin (5). The resulting signaling module acts as a bridge joining the appropriate receptor to the downstream effectors, enabling activation of the stress-activated MAPK.The most distal point at which signal and cell specificity for JNK and p38 activation is conferred is at the MAPKKK level. Mixed lineage kinases (MLKs) are a family of MAPKKKs activating JNK and p38. There are currently seven mammalian kinases belonging to the MLK family that have recently been reviewed (6, 7). These kinases can be divided into three subclasses based on sequence similarity and domain structure, and they include MLK1-4, dual leucine zipper kinases, and the zipper sterile ␣-motif kinases (ZAKs). Although much is known about the mechanisms re...