A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.     

Yeast MEK-dependent signal transduction: response thresholds and parameters affecting fidelity.

Ste7p and Mkk1p are MEK (MAPK/ERK kinase) family members that function in the mating and cell integrity signal transduction pathways in Saccharomyces cerevisiae. We selected STE7 and MKK1 mutations that stimulated their respective pathways in the absence of an inductive signal. Strikingly, serine-to-proline substitutions at analogous positions in Ste7p (position 368) and Mkk1p (position 386) were recovered by independent genetic screens. Such an outcome suggests that this substitution in other MEKs would exhibit similar properties. The Ste7p-P368 variant has higher basal enzymatic activity than Ste7p but still requires induction to reach full activation. The higher activity associated with Ste7p-P368 allows it to compensate for defects in the cell integrity pathway, but it does so only when it is overproduced or when Ste5p is missing. This behavior suggests that Ste5p, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7p specificity.     

Identification of a novel mitogen-activated protein kinase kinase activation domain recognized by the inhibitor PD 184352

Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 micro M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.     

Corticosteroid effects on lipid lateral diffusion in CEM-C1 and CEM-C7 acute lymphoblastic leukemia cells

We have examined glucocorticoid effects on CEM-C7 and CEM-C1 subclones of a leukemic human T-cell line using fluorescence photobleaching recovery techniques. Incubation with 10(-5) M triamcinolone acetonide (TA) increased lipid lateral diffusion on steroid-sensitive CEM-C7 cells but had no effect on steroid-resistant CEM-C1 cells. CEM-C7 cells incubated in serum-free medium responded only to TA but, when fetal calf serum was added to the incubation medium, would also respond to 10(-5) M dexamethasone and hydrocortisone. Thus, glucocorticoids can cause increased lipid lateral diffusion in CEM-C7 cells, while having no effect on steroid-resistant CEM-C1 cells.     

Spatial determinants of specificity in insulin action

Insulin is a potent stimulator of intermediary metabolism, however the basis for the remarkable specificity of insulin's stimulation of these pathways remains largely unknown. This review focuses on the role compartmentalization plays in insulin action, both in signal initiation and in signal reception. Two examples are discussed: (1) a novel signalling pathway leading to the phosphorylation of the caveolar coat protein caveolin, and (2) a recently identified scaffolding protein, PTG, involved directly in the regulation of enzymes controlling glycogen metabolism.