Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.

Fibroblast growth factor (FGF) activates a protein kinase cascade in SK‐N‐MC cells that regulates gene expression at a cyclic‐AMP response element (CRE) by stimulating the transcriptional activity of CREB. The activation of CREB is prevented by a dominant negative mutant of Ras and triggered via the same site (Ser133) that becomes phosphorylated in response to cyclic AMP and Ca2+. However, the effect of FGF is not mediated by cyclic AMP‐dependent protein kinase, TPA‐sensitive isoforms of protein kinase‐C, p70S6K or p90rsk (all of which phosphorylate CREB at Ser133 in vitro). Instead, we identify the FGF‐stimulated CREB kinase as MAP kinase‐activated protein (MAPKAP) kinase‐2, an enzyme that lies immediately downstream of p38 MAP kinase, in a pathway that is also stimulated by cellular stresses. We show that MAPKAP kinase‐2 phosphorylates CREB at Ser133 in vitro, that the FGF‐ or stress‐induced activation of MAPKAP kinase‐2 and phosphorylation of CREB and ATF‐1 are prevented by similar concentrations of the specific p38 MAP kinase inhibitor SB 203580, and that MAPKAP kinase‐2 is the only detectable SB 203580‐sensitive CREB kinase in SK‐N‐MC cell extracts. We also show that transfection of RK/p38 MAP kinase in SK‐N‐MC cells, but not transfection of p44 MAP kinase, activates Gal4‐CREB‐dependent transcription via Ser133. These findings identify a new growth factor and stress‐activated signaling pathway that regulates gene expression at the CRE.

Purpose: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non^small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLCassociated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. Experimental Design:We tested mutation-specific antibodies byWestern blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. Results: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry^based DNA sequencing. Conclusions: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.Lung cancer is a major cause of cancer-related mortality worldwide and is expected to remain a major health problem for the foreseeable future. Lung cancer is broadly divided into small-cell lung cancer (20% of lung cancers) and non -smallcell lung cancer (NSCLC; 80% of lung cancers). Somatic mutations in the epidermal growth factor receptor (EGFR) gene are found in a subset of NSCLC adenocarcinomas and are associated with sensitivity to the small-molecule EGFR tyrosine kinase inhibitors gefitinib (1, 2) and erlotinib (3). Different EGFR mutations have been reported, but the most common NSCLC-associated EGFR mutations are in-frame deletions in exon 19 (E746_A750del) and the point mutation replacing leucine with arginine at codon 858 in exon 21 (L858R; refs. 3 -5). These two mutations represent 85% to 90% of EGFR mutations in NSCLC patients. Data from clinical research have confirmed that patients with these mutations are highly responsive to EGFR inhibitors including gefitinib and erlotinib (5 -8).Based on these clinical findings, EGFR mutational analysis in lung adenocarcinoma may now be used to guide treatment decisions and to enroll patients in specific arms of clinical trials. Direct DNA sequencing of PCR-amplified genomic DNA has been developed to detect EGFR mutations i...

Adipose tissue is a metabolically responsive endocrine organ that secretes a myriad of adipokines. Antidiabetic drugs such as peroxisome proliferator-activated receptor (PPAR) ␥ agonists target adipose tissue gene expression and correct hyperglycemia via whole-body insulin sensitization. The mechanism by which altered gene expression in adipose tissue affects liver and muscle insulin sensitivity (and thus glucose homeostasis) is not fully understood. One possible mechanism involves the alteration in adipokine secretion, in particular the up-regulation of secreted factors that increase whole-body insulin sensitivity. Here, we report the use of transcriptional profiling to identify genes encoding for secreted proteins the expression of which is regulated by PPAR␥ agonists. Of the 379 genes robustly regulated by two structurally distinct PPAR␥ agonists in the epididymal white adipose tissue (EWAT) of db/db mice, 33 encoded for known secreted proteins, one of which was FGF21. Although FGF21 was recently reported to be up-regulated in cultured adipocytes by PPAR␥ agonists and in liver by PPAR␣ agonists and induction of ketotic states, we demonstrate that the protein is transcriptionally up-regulated in adipose tissue in vivo by PPAR␥ agonist treatment and under a variety of physiological conditions, including fasting and high fat diet feeding. In addition, we found that circulating levels of FGF21 protein were increased upon treatment with PPAR␥ agonists and under ketogenic states. These results suggest a role for FGF21 in mediating the antidiabetic activities of PPAR␥ agonists.

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The substrates of most protein kinases remain unknown because of the difficulty tracing signaling pathways and identifying sites of protein phosphorylation. Here we describe a method useful in detecting subclasses of protein kinase substrates. Although the method is broadly applicable to any protein kinase for which a substrate consensus motif has been identified, we illustrate here the use of antibodies broadly reactive against phosphorylated Ser/Thr-motifs typical of AGC kinase substrates. Phosphopeptide libraries with fixed residues corresponding to consensus motifs RXRXXT-*/S* (Akt motif) and S*XR (protein kinase C motif) were used as antigens to generate antibodies that recognize many different phosphoproteins containing the fixed motif. Because most AGC kinase members are phosphorylated and activated by phosphoinositide-dependent protein kinase-1 (PDK1), we used PDK1؊/؊ ES cells to profile potential AGC kinase substrates downstream of PDK1. To identify phosphoproteins detected using the Akt substrate antibody, we characterized the antibody binding specificity to generate a specificity matrix useful in predicting antibody reactivity. Using this approach we predicted and then identified a 30-kDa phosphoprotein detected by both Akt and protein kinase C substrate antibodies as S6 ribosomal protein. Phosphospecific motif antibodies offer a new approach to protein kinase substrate identification that combines immunoreactivity data with protein data base searches based upon antibody specificity.

Although activation of protein kinase C (PKC) is known to promote cell survival and protect against cell death, the PKC targets and pathways that serve this function have remained elusive. Here we demonstrate that two potent activators of PKC, 12-O-tetradecanoylphorbol-13-acetate and bryostatin, both stimulate phosphorylation of Bad at Ser(112), a site known to regulate apoptotic cell death by interleukin-3. PKC inhibitors but not PI 3-kinase/Akt inhibitors block 12-O-tetradecanoylphorbol-13-acetate-stimulated Bad phosphorylation. PKC isoforms tested in vitro were unable to phosphorylate Bad at Ser(112), suggesting that PKC acts indirectly to activate a downstream Bad kinase. p90(RSK) and family members RSK-2 and RSK-3 are activated by phorbol ester and phosphorylate Bad at Ser(112) both in vitro and in vivo. p90(RSK) stimulates binding of Bad to 14-3-3 and blocks Bad-mediated cell death in a Ser(112)-dependent manner. These findings suggest that p90(RSK) can function in a PKC-dependent pathway to promote cell survival via phosphorylation and inactivation of Bad-mediated cell death.

A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that kaempferol may have cancer chemopreventative properties. However, the precise underlying protective mechanisms are poorly understood. To elucidate these mechanisms, we challenged human lung cancer cell line A549 with kaempferol and investigated its effects upon cellular growth and signal transduction pathways. Treatment of A549 cells with kaempferol resulted in a dose- and time-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 0.9+/-0.5, 5.2+/-1.5, 16.8+/-2.0, 25.4+/-2.6, and 37.8+/-4.5% on treatment with 0, 17.5, 35.0, 52.5, and 70.0 microM kaempferol, respectively. Concomitantly, kaempferol treatments led to a 1.2-, 2.7-, 3.3-, and 3.4-fold increase in Bax. Similar elevations were also observed in Bad which increased 1.2-, 3.3-, 3.7-, and 4.7-fold, respectively, as compared to control. Bcl-2 and Bcl-xL expression were inhibited in a dose-dependent fashion. While the Akt-1 and phosphorylated Akt-1 were inhibited, the mitogen-activated protein kinase (MAPK) was activated upon kaempferol treatment. Kaempferol induced apoptosis was associated with the cleavage of caspase-7 and poly ADP-ribose polymerase (PARP). Inhibition of MEK1/2 but not PI-3 kinase blocked kaempferol-induced cleavage of caspase-7, PARP cleavage, and apoptosis. The results suggest that inactivation of Akt-1 and alteration of Bcl-2 family of proteins are not sufficient for kaempferol to induce apoptosis and activation of MEK-MAPK is a requirement for kaempferol-induced cell death machinery in A549 cells.