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Recently, we reported the synthesis and use of [32P]2-azido-NAD+ as a probe to study the structural organization of G-proteins. Pertussis toxin was used to 'tether' [32P]2-azido-ADP-ribose of [32P]2-azido-NAD+ to Cys347 of the alpha subunit of the G-protein Gt. Light activation of the azide moiety covalently cross-linked the domain containing Cys347 at the C-terminus of alpha t with neighbouring intra- and inter-molecular domains of holo-transducin. The radiolabel from [32P]2-azido-ADP-ribose was then transferred to the 'acceptor' domain by cleaving the thioglycosidic bond between Cys347 and [32P]2-azido-ADP- ribose with mercuric acetate. ADP-ribosylation followed by photocross-linking of holo-transducin indicated intramolecular interactions of the C-terminal domain with other alpha t domains and intermolecular interactions with holotransducin alpha and gamma subunits. The radiolabelled peptides, which were radiolabelled because of the transfer of the photoactive moiety, were identified by utilizing 2-(2'-nitrophenylsulphenyl)-3-methyl-3'- bromoindolenine ('BNPS-skatole') and CNBr. The results indicate that the C-terminus of alpha t interacts with both N-terminal and C-terminal domains within the alpha t molecular. Mapping the interacting sites between cross-linked alpha dimers and alpha trimers indicates that the C-terminal domain of alpha t is involved in the formation of alpha t homopolymers in solution. In addition, our studies place the beta gamma subunit in close proximity to Cys347 of alpha t, as indicated by the transfer of [32P]2-azido-ADP-ribose from Cys347 to the gamma subunit, which was further localized to the C-terminal half of gamma t. The studies presented here identify the C-terminal intra- and inter-molecular interactions of the alpha subunit of holo-transducin.

Sequential protein kinase reactions involve the phosphorylation and activation of multiple kinases in a pathway. The growth factor receptor tyrosine kinase regulation of the mitogen-activated protein kinase (MAPK) pathway was defined in 1993. The MAPK pathway involves sequential protein kinase reactions. Notable advances were made in defining tyrosine kinase receptor regulation of Ras, and these discoveries were combined with the identification of Raf-1, a serine-threonine protein kinase in the MAPK pathway, as an effector for Ras GTP.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to a variety of extracellular stimuli in many different cell types. The kinases that activate MAPK, the MAPK/ERK Kinases (MEKs), are also activated by phosphorylation. We have studied the influence of specific oncogenes on the regulation of MEK activity in NIH3T3 and Rat1a fibroblasts. We show that a similar MEK activity phosphorylates and activates MAPK in both growth factor-stimulated (epidermal growth factor and thrombin) and oncogene (gip2, v-src, and v-raf)-transfected cells. Gip2 and v-Src activated MEK-1 in transfected Rat 1a cells, whereas v-Raf activated MEK-1 in transfected NIH3T3 cells. These cell-selective differences in MEK activation parallel constitutive MAPK activation in these cell lines. Stable expression of the v-ras oncogene resulted in little constitutive MEK activation in either cell line, even though both were highly transformed. The growth factor and oncoprotein regulated MEK activity co-fractionated by Mono S chromatography with the 45-kDa MEK-1 protein. We further demonstrate in NIH3T3 and Rat 1a cells that Raf-1 is activated, as measured by its ability to phosphorylate MEK-1, in response to epidermal growth factor but not thrombin. Thus, the regulatory network of protein kinases that activate MAPK converges at MEK but diverges with the kinases that phosphorylate and activate MEK.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.

A radioactive and photoactivatable derivative of NAD+, 2-azido-[adenylate-32P]NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-[adenylate-32P]ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-[adenylate-32P]ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.