We have deduced the entire 1,370-amino-acid sequence of the human insulin receptor precursor from a single complementary DNA clone. The precursor starts with a 27-amino-acid signal sequence, followed by the receptor alpha-subunit, a precursor processing enzyme cleavage site, then the beta-subunit containing a single 23-amino-acid transmembrane sequence. There are sequence homologies to human epidermal growth factor receptor and the members of the src family of oncogene products.

The RAS-mitogen activated protein kinase (MAPK) signalling pathway has long been viewed as an attractive pathway for anticancer therapies, based on its central role in regulating the growth and survival of cells from a broad spectrum of human tumours. Small-molecule inhibitors designed to target various steps of this pathway have entered clinical trials. What have we recently learned about their safety and effectiveness? Will the MAPK pathway prove amenable to therapeutic intervention?

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

BAD is a distant member of the Bcl-2 family that promotes cell death. Phosphorylation of BAD prevents this. BAD phosphorylation induced by interleukin-3 (IL-3) was inhibited by specific inhibitors of phosphoinositide 3-kinase (PI 3-kinase). Akt, a survival-promoting serine-threonine protein kinase, was activated by IL-3 in a PI 3-kinase-dependent manner. Active, but not inactive, forms of Akt were found to phosphorylate BAD in vivo and in vitro at the same residues that are phosphorylated in response to IL-3. Thus, the proapoptotic function of BAD is regulated by the PI 3-kinase-Akt pathway.

CI-1040 was generally well tolerated but demonstrated insufficient antitumor activity to warrant further development in the four tumors tested. PD 0325901, a second generation MEK inhibitor, has recently entered clinical development and, with significantly improved pharmacologic and pharmaceutical properties compared with CI-1040, it may better test the therapeutic potential of MEK inhibition in cancer.

CI-1040 was well tolerated at 800 mg BID administered with food. Both target suppression and antitumor activity were demonstrated in this phase I study.

Preparations of insulin receptor from cultured 3T3-L1 adipocytes and human placenta previously was found to catalyze the phosphorylation of the 90,000-dalton component of the insulin receptor on tyrosine residues. This insulin-dependent phosphorylation has now been shown to coincide with the generation of an activated, insulin-independent, receptor protein kinase. Activation is dependent upon ATP, divalent cations (Mg2' and Mn2+), and insulin (half-maximal activation occurs at 6-8 nM insulin). The time required for activation is consistent with that needed for insulin-dependent self-phosphorylation of the receptor present in eluates from wheat germ lectin-agarose columns and in preparations of affinity-purified placental receptor. Activation proceeds unabated in the presence of soybean trypsin inhibitor at 0.1 mg/ml and the activated, insulin-independent, protein kinase sediments in 5-20% sucrose gradients at the same position as the unmodified receptor. Under steady-state conditions, the phosphorylated receptor binds insulin in the same fashion as the unmodified receptor. it is proposed that the self-phosphorylated form of the receptor is the insulin-activated protein kinase that catalyzes the phosphorylation of exogenous protein and peptide substrates. A corollary of this hypothesis is that enzymatic dephosphorylation may be essential for reversibly terminating the activity of the insulin-receptor protein Idnase.Insulin activates a protein kinase that catalyzes the phosphorylation of the insulin receptor in intact cells (1-3) and in preparations of purified insulin receptor (4). In vitro, the insulin receptor derived from cultured 3T3-L1 adipocytes and human placenta is phosphorylated on tyrosine residues in the 90,000-dalton subunit of the receptor (3, 5). Evidence that insulin-dependent tyrosine kinase activity copurifies with the receptor to near-homogeneity suggests that the receptor itself is the protein kinase. However, it is not known which of its subunits possesses this activity. All of the tyrdsine protein kinases that have been described thus far undergo self-phosphorylation. This modification, however, has not been reported to affect enzyme function.We have now found that incubation of the insulin receptor with insulin, ATP, and divalent cations under conditions in which it becomes phosphorylated stimulates its subsequent ability to catalyze the phosphorylation of exogenous substrates. The phosphorylated receptor remains fully active after gel filtration at pH 6.0, a procedure which restores insulin dependency to the insulin-treated, unphosphorylated receptor.MATERIALS AND METHODS Materials. To prepare insulin receptor, cultured 3T3-L1 adipocytes were grown and induced to differentiate as described (6). A glycoprotein-rich fraction derived from solubilized membranes of either human placenta or 3T3-L1 adipocytes was prepared by wheat germ agglutinin-agarose chromatography as described (3). This procedure purified the insulin receptor approximately 15-fold from the solubilized membranes and c...

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