Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.     

Differential Inhibitor of G�¦� Signaling to AKT and ERK Derived from Phosducin-like Protein: EFFECT ON SPHINGOSINE 1-PHOSPHATE-INDUCED ENDOTHELIAL CELL MIGRATION AND IN VITRO ANGIOGENESIS*

Differential inhibitors of Gβγ-effector regions are required to dissect the biological contribution of specific Gβγ-initiated signaling pathways. Here, we characterize PhLP-M1-G149, a Gβγ-interacting construct derived from phosducin-like protein 1 (PhLP) as a differential inhibitor of Gβγ, which, in endothelial cells, prevented sphingosine 1-phosphate-induced phosphorylation of AKT, glycogen synthase kinase 3β, cell migration, and tubulogenesis, while having no effect on ERK phosphorylation or hepatocyte growth factor-dependent responses. This construct attenuated the recruitment of phosphoinositide 3-kinase γ (PI3Kγ) to the plasma membrane and the signaling to AKT in response to Gβγ overexpression. In coimmunoprecipitation experiments, PhLP-M1-G149 interfered with the interaction between PI3Kγ and Gβγ. Other PhLP-derived constructs interacted with Gβγ but were not effective inhibitors of Gβγ signaling to AKT or ERK. Our results indicate that PhLP-M1-G149 is a suitable tool to differentially modulate the Gβγ-initiated pathway linking this heterodimer to AKT, endothelial cell migration, and in vitro angiogenesis. It can be also useful to further characterize the molecular determinants of the Gβγ-PI3Kγ interaction.Heterotrimeric G protein signaling depends on the actions of GTP-loaded Gα and free Gβγ, the two functional components of the heterotrimer, leading to the generation of second messengers and cell specific functional events (1, 2). Differential inhibitors of Gβγ are required to dissect the biological impact of different Gβγ-dependent effectors. Gβγ actions can be blocked by competition with peptides derived from its effectors. For example, the effect of Gβγ on adenylyl cyclase II, G protein-activated inward rectifier K⁺ channel, G protein-coupled receptor kinase 2, and phospholipase Cβ3, is attenuated by a peptide from adenylyl cyclase II (3). In addition, RACK1 (receptor for activated C kinase 1) selectively inhibits the effect the chemokine receptor CXCR2 on the activation of phospholipase Cβ2 and adenylyl cyclase II in HEK293 cells, without affecting other functions of Gβγ (4). Recently, Smrcka and colleagues characterized the effect of small molecule inhibitors of Gβγ, suggesting their potential application in therapeutic strategies targeting particular Gβγ-dependent pathways (5). Emerging possibilities to target this heterodimer in pathological situations such as inflammation and angiogenesis are based on the role of Gβγ in cell survival and chemotaxis. To the best of our knowledge, no molecular tool is yet available to differentially inhibit Gβγ signaling to AKT.³Gβγ is a key transducer of sphingosine 1-phosphate (S1P)-elicited angiogenic signals promoting endothelial cell migration, proliferation, and survival (6–12). Multiple Gβγ-dependent effectors are potentially involved in the molecular events required for endothelial cell migration. These include lipid kinases such as PI3Kγ and PI3Kβ (13), and a novel family of Rac guanine nucleotide exchange factors, represented by P-REX1, which is activated by Gβγ and phosphatidylinositol 3,4,5-trisphosphate (14–16). Gβγ signaling is frequently attributed to pertussis toxin-sensitive G_i coupled receptors, and it has been consistently revealed by the antagonistic effect of the carboxyl-terminal region of G protein-coupled receptor kinase 2, which sequesters Gβγ thereby inhibiting all its intracellular actions (17). In addition, mutational analysis of Gβ revealed that different residues, all of them mapping to the interface of contact between Gβγ and Gα, are important for the activation of distinct Gβγ effector molecules (18).Phosducin was originally identified as a phosphoprotein restricted to the retina and pineal gland forming a complex with Gβγ (19, 20). It was considered a protein kinase A-sensitive regulator of G protein-mediated signaling (21, 22). Further studies identified a family of phosducin-like proteins (PhLPs) (23, 24). Phosducin and Gα share affinity for the same region of Gβγ, as revealed by the structural analysis of Gβγ in complex with Gα or phosducin and by in vitro binding experiments (25). This area of interaction includes some of the residues considered necessary for the activation of Gβγ-dependent effectors (18, 26). It was initially postulated that phosducin and related proteins, by interfering with the availability of free Gβγ, exert an inhibitory role on Gβγ signaling. However, recent genetic evidence raised an apparently conflicting situation; the knockout of PhLP in fungi resulted in a phenotype equivalent to the absence of Gβγ, contrary to its expected role as an inhibitor (27). Novel experimental evidence indicated that PhLP has a positive effect on Gβγ signaling due to its participation in the assembly of the heterodimer, helping to stabilize free Gβ subunits leaving the ribosome after synthesis (28–31).Despite the positive role of full-length PhLP in the assembly of Gβγ heterodimers, it is still possible that different fragments of this protein, which could retain their interaction with distinct regions of Gβγ, might function as inhibitors of Gβγ signaling. Accordingly, we characterized here the effect of different PhLP-derived constructs on the signaling pathways elicited by S1P or HGF in endothelial cells. In addition, we explored the mechanism by which PhLP-M1-G149 interferes with Gβγ preventing the activation of AKT.     

Decreased angiotensin II binding affinity and binding capacity in the anterior pituitary gland of adult spontaneously hypertensive rats

Angiotensin II (ANG) binding sites were quantified in single pituitary glands from 4-week-old and 14-week-old male spontaneously hypertensive rats (SHR) and age-matched male normotensive Wistar-Kyoto (WKY) control rats after incubation with 125I-[Sar1]-ANG, autoradiography with computerized densitometry, and comparison to 125I-standards. The maximum binding capacity (Bmax) decreased while the dissociation constant (Kd) for ANG increased in 14-week-old SHR when compared to age-matched WKY control rats (Bmax: 265 +/- 9 and 224 +/- 4 fmol/mg protein; Kd: 0.79 +/- 0.04 and 1.14 +/- 0.08 10(-9) M in WKY and SHR, respectively). Conversely, no difference between rat strains was found in 4-week-old animals. Our results suggest that pituitary ANG binding sites may play a role in the pathophysiology of established genetic hypertension.     

Atrial natriuretic peptide receptors in sympathetic ganglia: biochemical response and alterations in genetically hypertensive rats

High concentration of atrial natriuretic peptide (99-126) (ANP) receptors were localized by quantitative autoradiography in superior cervical and stellate ganglia from young and adult Wistar Kyoto (WKY) rats. ANP increased cyclic GMP formation in stellate ganglia from adult rats. Both young and adult spontaneously hypertensive rats (SHR) had a much lower number of ANP receptors in the sympathetic ganglia. In spite of low receptor concentration, the cyclic GMP response to ANP in SHR was unchanged. These results suggest the existence of physiologically active ANP receptors in the rat sympathetic ganglia. These receptors may also be involved in the pathophysiology of spontaneous hypertension.     

Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated

It has long been known, from the distribution of multiple amino acid replacements, that not all amino acids of a sequence are replaceable. More recently, the phenomenon was observed at the nucleotide level in mitochondrial DNA even after allowing for different rates of transition and transversion substitutions. We have extended the search to globin gene sequences from various organisms, with the following results: (1) Nearly every data set showed evidence of invariable nucleotide positions. (2) In all data sets, substitution rates of transversions and transitions were never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only rarely (e.g., the third codon position of beta hemoglobins) was it possible to fit the data set solely by making allowance for the number of invariable positions and for the relative rates of transversion and transition substitutions. (4) For one data set (the second codon position of beta hemoglobins) we were able to simulate the observed data by making the allowance in (3) and having the set of covariotides (concomitantly variable nucleotides) be small in number and be turned over in a stochastic manner with a probability that was appreciable. (5) The fit in the latter case suggests, if the assumptions are correct and at all common, that current procedures for estimating the total number of nucleotide substitutions in two genes since their divergence from their common ancestor could be low by as much as an order of magnitude. (6) The fact that only a small fraction of the nucleotide positions differ is no guarantee that one is not seriously underestimating the total amount of divergence (substitutions). (7) Most data sets are so heterogeneous in their number of transition and transversion differences that none of the current models of nucleotide substitution seem to fit them even after (a) segregation of coding from noncoding sequences and (b) splitting of the codon into three subsets by codon position. (8) These frequently occurring problems cannot be seen unless several reasonably divergent orthologous genes are examined together.      

Diverse biologic properties imparted by the c-fgr proto-oncogene.

The c-fgr proto-oncogene specifies a nonreceptor protein-tyrosine kinase, p55c-fgr, a member of the src family. In the present study, we have mutagenized c-fgr to mimic alterations found at the 3' end of the v-fgr oncogene and have investigated the biologic effects of normal and mutant p55c-fgr expression. Genes lacking 10 or 13 codons at the 3' end, as well as a gene encoding phenylalanine instead of tyrosine at codon 523, were potent oncogenes when transfected into NIH 3T3 cells. Specific enzymatic activities of the more highly transforming gene products were 3-4-fold greater than that of p55c-fgr. In vivo, the amount of tyrosine phosphorylation of cellular proteins was directly proportional to potency in focus-forming assays. These findings are the first to identify highly transforming mutations of the c-fgr proto-oncogene. The proto-oncogene was also active in transforming assays, demonstrably greater than that of a kinase-deficient mutant. Foci arising in c-fgr-transfected cultures expressed abundant enzyme that was normal by a number of criteria. In addition, growth rates for cells expressing p55c-fgr were restricted, as compared with cells expressing a kinase-deficient protein or cells transformed by proteins with high specific enzymatic activities. Thus, enzymatically active p55c-fgr can simultaneously activate transforming and growth inhibitory pathways.     

Activation of transforming G protein-coupled receptors induces rapid tyrosine phosphorylation of cellular proteins, including p125FAK and the p130 v-src substrate.

We have used the family of human muscarinic receptors (mAChRs) as a model for receptors coupled to G proteins and have shown that genes for certain mAChR subtypes can behave as potent agonist-dependent oncogenes. Furthermore, transforming mAChRs can transduce mitogenic signals in transfected NIH 3T3 cells. In this study, we show that in cells expressing ml mAChRs, the cholinergic agonist carbachol, induces a rapid and dose-dependent increase in tyrosine phosphorylation of cellular proteins which are different from those induced by PDGF. Interestingly, carbachol, but not PDGF, induces an increase in tyrosine phosphorylation of the p125FAK and p130 v-src substrates. Thus, growth promoting pathways activated by receptors coupled to G proteins might involve tyrosine phosphorylation of a small set of cellular proteins previously identified as substrates for oncogene-encoded tyrosine kinases.