Geranylgeranylation signals to the Hippo pathway for breast cancer cell proliferation and migration.

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration.

Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-.

To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location.

A surface-exposed region of G(salpha) in which substitutions decrease receptor-mediated activation and increase receptor affinity.

The mechanism by which receptors activate G proteins is unclear because a connection between the receptor and the nucleotide binding site has not been established. To investigate this mechanism, we evaluated the roles in receptor interaction of three potential receptor contact sites in alpha(s): the alpha2/beta4, alpha3/beta5, and alpha4/beta6 loops. Substitutions of alpha(i2) homologs for alpha(s) residues in the alpha2/beta4 loop and alanine substitutions of residues in the alpha4/beta6 loop do not affect activation by the beta(2)-adrenergic receptor. However, replacement of five alpha(s) residues in the alpha3/beta5 loop region with the homologous alpha(i2) residues decreases receptor-mediated activation of alpha(s) and increases the affinity of G(s) for this receptor. The substitutions do not alter guanine nucleotide binding or hydrolysis, or activation by aluminum fluoride, indicating that the effects on receptor interaction are not due to a destabilization of the guanine-nucleotide bound state. In a model of the receptor-G protein complex, the alpha3/beta5 loop maps near the second and third intracellular loops of the receptor. The effects of the alpha3/beta5 substitutions suggest that the wild-type residues may be receptor contact sites that are optimized to ensure the reversibility of receptor-G protein interactions. Furthermore, the alpha3/beta5 region corresponds to an exchange factor contact site in both EF-Tu and Ras, suggesting that the mechanisms by which seven-transmembrane receptors and exchange factors catalyze nucleotide exchange may share common elements.

cAMP regulates Ca2+-dependent exocytosis of lysosomes and lysosome-mediated cell invasion by trypanosomes.

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a beta-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+]i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.

Mutations at the domain interface of GSalpha impair receptor-mediated activation by altering receptor and guanine nucleotide binding.

G protein alpha subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous alphai2 residues for four alphas residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of alphas to be activated by the beta-adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of alphas for the beta-adrenergic receptor, based on an increased amount of high affinity binding of the beta-adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the beta-adrenergic receptor to increase the apparent affinity of alphas for the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous alphai2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.

Receptor-mediated activation of Gsalpha: evidence for intramolecular signal transduction.

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of alphas (residues 236-394) to identify residues that play a role in receptor-mediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc- S49 lymphoma cells, which lack endogenous alphas. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an alpha helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding, are positioned to relay the receptor-initiated signal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of alphas with that of alphai2 in an alphas/alphai2/alphas chimera corrects the defect in receptor-mediated activation caused by alphai2 substitutions on the GTPase side of the interface. Thus, complementary interactions between residues across the domain interface seem to play a role in receptor-catalyzed activation.

Identification of common and distinct residues involved in the interaction of alphai2 and alphas with adenylyl cyclase.

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.

Localization of the effector-specifying regions of Gi2alpha and Gqalpha.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain. An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP. Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.

Identification of effector-activating residues of Gs alpha.

The heterotrimeric G proteins transduce extracellular signals by interacting with specific intracellular effectors. We have used a scanning mutagenesis approach to identify amino acids of alpha S, the alpha subunit of Gs, that determine the specificity of its interaction with its effector, adenylyl cyclase. In alpha subunit chimeras, residues 236-356 of alpha S comprise the shortest linear stretch that is required for activation of adenylyl cyclase. Within these 121 residues, we identified four clusters of residues in which substitutions prevented effector activation. Mutations in three of these regions did not affect alpha subunit expression or the GTP-induced conformational change. The identified alpha S residues in the NH2-terminal half of the 121-residue region endowed the cognate alpha i2 segment with the ability to activate effector, while those in the COOH-terminal half did not. In a three-dimensional G alpha model, based on the structure of p21ras, the effector-activating residues of alpha S form a surface on the membrane-facing side of the molecule; this surface includes a region that changes conformation upon binding GTP.

Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation.

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.

Expression in Escherichia coli of a functional Dictyostelium myosin tail fragment.

The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity.

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.

Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium.

Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [32P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a gamma-[32P]ATP-containing reaction mixture.

Affinity labeling and partial purification of nerve growth factor receptors from rat pheochromocytoma and human melanoma cells.

A cell surface glycoprotein receptor for nerve growth factor (NGF) has been identified by covalent crosslinking to 125I-labeled NGF (125I-NGF). Either ethyldimethylisopropyl-aminocarbodiimide or hydroxysuccinimidyl-p-azidobenzoate causes highly specific crosslinking of 125I-NGF to a similar receptor species on rat pheochromocytoma PC12 cells and on human melanoma A875 cells. The NGF-receptor complex migrates as a broad band in NaDodSO4/polyacrylamide gel electrophoresis with an apparent Mr of approximately equal to 100,000. Because the NaDodSO4-denatured complex apparently contains a single Mr 13,000 NGF chain, the apparent molecular weight of the receptor itself is 87,000. Inhibition of protein glycosylation by tunicamycin generates smaller 125I-NGF-receptor complexes. The mobility of the smallest of these in NaDodSO4 gel electrophoresis corresponds to a Mr of 90,000 for the complex and, hence, 77,000 for the carbohydrate-deficient receptor. A second NGF-receptor complex with a Mr of 225,000 also is obtained from A875 cells but only occasionally from PC12 cells. Tunicamycin treatment decreases the molecular weight of these species by 10,000-15,000. Substantial purification of the Mr 100,000 NGF-receptor complex was achieved by lectin affinity chromatography on columns of wheat germ agglutinin linked to Affi-Gel 15. The specific absorption of NGF-receptor complexes to these columns indicates that the receptor is a glycoprotein that contains N-acetyl-D-glucosamine.