Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP‐binding protein Ras and then the protein kinase p74raf‐1. p74raf‐1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf‐1. This represents the first characterization of sites phosphorylated by this proto‐oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A ‘kinase‐dead’ MAPKK1 mutant was phosphorylated at the same residues as the wild‐type enzyme, establishing that both sites are phosphorylated directly by p74raf‐1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf‐1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.

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ERBB3, a member of the epidermal growth factor receptor (EGFR) family, is unique in that its tyrosine kinase domain is functionally defective. It is activated by neuregulins, by other ERBB and nonERBB receptors as well as by other kinases, and by novel mechanisms. Downstream it interacts prominently with the phosphoinositol 3-kinase/AKT survival/mitogenic pathway, but also with GRB, SHC, SRC, ABL, rasGAP, SYK and the transcription regulator EBP1. There are likely important but poorly understood roles for nuclear localization and for secreted isoforms. Studies of ERBB3 expression in primary cancers and of its mechanistic contributions in cultured cells have implicated it, with varying degrees of certainty, with causation or sustenance of cancers of the breast, ovary, prostate, certain brain cells, retina, melanocytes, colon, pancreas, stomach, oral cavity and lung. Recent results link high ERBB3 activity with escape from therapy targeting other ERBBs in lung and breast cancers. Thus a wide and centrally important role for ERBB3 in cancer is becoming increasingly apparent. Several approaches for targeting ERBB3 in cancers have been tested or proposed. Small inhibitory RNA (siRNA) to ERBB3 or AKT is showing promise as a therapeutic approach to treatment of lung adenocarcinoma.

We have investigated the relationship between hydrolysis of phosphatidylcholine (PC)

NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database revealed high sequence identity of the product of this gene to serinethreonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identity was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity.The short arm of human chromosome 3 harbors a number of tumor suppressor genes. Two of these genes were cloned and characterized: the von Hippel-Lindau disease gene at 3p25 (33) and the hereditary nonpolyposis colorectal cancer gene at 3p21-p22 (6, 42). At least three other tumor suppressor loci are considered to be located on 3p: 3p25, which is frequently deleted in breast cancers (8); the 3p21 region, which is involved in the origin and/or development of small cell lung cancer (SCLC) and non-SCLC (4), testicular (37), ovarian (56), and renal cell (29, 63) carcinomas; and the 3p12-p13 region, which shows loss of heterozygosity or homozygous deletions of markers in several cases of breast cancer (8), SCLC (34,44), and renal cell carcinoma (38). In the case of SCLC, heterozygous deletions of large segments of 3p are frequently observed, complicating the definition of the tumor suppressor locus. Recently homozygous deletions have been found in several SCLC-derived cell lines (13, 28), defining the putative SCLC gene locus to 3p21.3. At the same time, a 2-Mb DNA fragment from the 3p21-22 region, overlapping with the SCLC cell line NCI-H740 deletion, was shown to suppress tumorigenicity in nude mice when transfected into a mouse fibrosarcoma cell line (26). In the present study, NotI linking clones, which are markers of human genes (1), were used to identify transcribed sequences within the large NCI-H740 deletion. One of the genes identified within this region codes for a sequence which has extensive homology to the previously identified gene for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2).MAPKAP kinase 2 was originally isolated from rabbit skeletal muscle (50). In vitro studies have shown that the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) isoforms are the only detectable MAPKAP kinase 2-reactivating enzymes in sk...

The ErbB3 receptor and the downstream signaling kinase Akt are implicated in proliferation of lung adenocarcinoma cells. Inhibition by siRNAs to ErbB3 and Akt isoforms 1, 2 and 3 was utilized to investigate the contribution of these molecules to tumor survival, spreading and invasiveness, and the roles of specific Akt isoforms. ErbB3 siRNA stably and dose-dependently suppressed ErbB3 protein for 2 days or more, and reduced cell numbers, by both suppressing cell cycle and causing apoptosis and necrosis. It also inhibited soft agar growth, cell motility and migration, and invasiveness. Akt1, 2 and 3 siRNAs had similar suppressive effects on cell number, apoptosis/ necrosis and soft agar growth. However, although Akt1 siRNA had no effect on cell migration or invasion, Akt2 siRNA effectively suppressed both activities, and Akt3 siRNA had moderate effectiveness. In A549 cells, ErbB3 is indicated as having major effects on cell division, survival, motility, migration and invasiveness. All three Akt isoforms are to varying degrees involved in these cell behaviors, with Akt2 especially implicated in migration and invasion. ErbB3 and the Akts are promising targets for therapy, and siRNAs may be useful for this purpose.

Although ErbB3, a member of the epidermal growth factor receptor family, has been implicated in mammary tumorigenesis, investigation of its role in lung tumorigenesis has been limited. We found that ErbB3 was present at high levels in five of seven human lung adenocarcinoma cell lines examined, along with its ligands, heregulins alpha and beta, whereas ErbB3 was absent from HPL1D, a non- transformed cell line from human pulmonary peripheral epithelium. Interactions and effects of ErbB3 were studied in detail in adenocarcinoma lines H441 and H1373. Complexes containing phosphorylated ErbB2, phosphorylated ErbB3 and the p85 regulatory subunit of phosphoinositidyl 3-kinase were detected by co-immunoprecipitation experiments and were present constitutively even in the absence of serum-stimulated cell division. Serum treatment increased the pErbB3/p85 complexes and also stimulated phosphorylation of Akt and GSK3beta, increase in cyclin D1 and cell cycle progression, and these events were blocked by the Akt activation inhibitor LY294002. An ErbB3-specific antisense oligonucleotide reduced amounts of ErbB3 protein and p85 complex in both cell lines, and significantly suppressed cell proliferation. These results together suggest involvement of ErbB3 in growth of lung adenocarcinomas, through activation of phosphoinositidyl 3 kinase and Akt, inactivation of GSK3beta and stabilization of cyclin D1 for cell cycle maintenance. It could be a useful therapeutic target.

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-␤ (GSK3-␤), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/ nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-␣ (TGF-␣) and responded to TGF-␣ stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-␤, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-␣ were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-␣ activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/ Gab1/Shp2 after TGF-␣ stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.Keywords: EGFR; ErbB3; Gab1; Akt; lung epithelial cells Complex interactions of growth factors, receptors, and signaling pathways regulate normal cell division of lung epithelial cells and, in their derangement, contribute to the development of malignancy. Systematic distinction of essential from secondary changes is facilitated by the availability of two pairs of cell lines derived from cells of the peripheral lung epithelium of BALB/c female mice, which grew out after explanting the lungs (1, 2). Each of these pairs was derived from a single immortalized cell line, one subline of which underwent spontaneous malignant transformation to an adenocarcinoma line showing growth in soft agar and as nude mouse xenografts. These pairs are designated E9/E10 and A5/C10, where E9 and A5 are the malignant lines.The presence of lamellar bodies and cytoskeletal features indicated that type II cells may have been the origin of these lines (1, 2), confirmed by specific staining with antibodies to cytokeratins (3). We have confirmed lamellar body-like struc- tures in E10 and C10 cells by electron microscopy (unpublished data). E10 and C10 present 2-5% S-phase cells at confluence, compared with 20-30% of E9 and A5 cells (3). The two nontransformed cell lines, compared with the two transformed lines, have higher levels of fibronectin, laminin, and vitronectin; more organized cytoskeletons; more numerous gap junctions; and more glucocorticoid-and platelet-derived growth factor receptors (3).We have used these paired lines to investigate mechanisms of regulation of cell growth via the epidermal growth factor receptor (EGFR) ErbB1 in malignant compared with nontransforme...