ABSTRACT
Endocytosis is differentially regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) and phospholipase D (PLD). However, the relationship between HIF-1alpha and PLD in endocytosis is unknown. HIF-1alpha is degraded through the prolyl hydroxylase (PHD)/von Hippel-Lindau (VHL) ubiquitination pathway in an oxygen-dependent manner. Here, we show that PLD1 recovers the decrease in epidermal growth factor receptor (EGFR) endocytosis induced by HIF-1alpha independent of lipase activity via the Rab5-mediated endosome fusion pathway. EGF-induced interaction of PLD1 with HIF-1alpha, PHD and VHL may contribute to EGFR endocytosis. The pleckstrin homology domain (PH) of PLD1 itself promotes degradation of HIF-1alpha, then accelerates EGFR endocytosis via upregulation of rabaptin-5 and suppresses tumor progression. These findings reveal a novel role of the PLD1-PH domain as a positive regulator of endocytosis and provide a link between PLD1 and HIF-1alpha in the EGFR endocytosis pathway.
Subject(s)
Animals , Female , Humans , Blood Proteins/chemistry , Endocytosis , HEK293 Cells , HT29 Cells , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice, Nude , Neoplasms/genetics , Phospholipase D/chemistry , Phosphoproteins/chemistry , Protein Structure, Tertiary , ErbB Receptors/metabolism , Signal Transduction , Up-Regulation , Vesicular Transport Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of Rab5a and APPL1 in breast cancer and fibroma, and analyze their correlation with HER-2 expression, metastasis and development of breast cancer.</p><p><b>METHODS</b>Rab5a and APPL1 in paraffin embedded tissues of 74 breast carcinomas and 40 breast fibromas were detected by immunohistochemistry. Their relationship with metastasis, pathological grade, and HER-2 expression in breast cancer was determined by statistical analysis.</p><p><b>RESULTS</b>There was no expression or low expression of Rab5a and APPL1 in the breast fibroma, but the positive expression rate of Rab5a and APPL1 in the breast carcinomas were 91.9% and 83.8%, respectively. No significant difference in expression of Rab5a and APPL1 was found between metastatic and non-metastatic groups, and pathological grade I/II and grade III groups. But Rab5a was overexpressed in HER-2-positive group compared with that in the HER-2-negative group.</p><p><b>CONCLUSIONS</b>Rab5a and APPL1 are overexpressed in breast cancer, and are positively correlated with the HER-2 expression. These proteins may influence the growth and proliferation of breast cancer cells by HER-2 signal transduction.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Young Adult , Adaptor Proteins, Signal Transducing , Metabolism , Breast Neoplasms , Metabolism , Pathology , Carcinoma, Ductal, Breast , Metabolism , Pathology , Carcinoma, Intraductal, Noninfiltrating , Metabolism , Pathology , Carcinoma, Squamous Cell , Metabolism , Pathology , Fibroma , Metabolism , Pathology , Immunohistochemistry , Lymphatic Metastasis , Neoplasm Grading , Paraffin Embedding , Receptor, ErbB-2 , Metabolism , rab5 GTP-Binding Proteins , MetabolismABSTRACT
Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.
Subject(s)
Animals , Cattle , Female , Humans , Male , Rats , Amino Acid Sequence , Cell Membrane , Metabolism , Radiation Effects , Conserved Sequence , Cyclic Nucleotide-Gated Cation Channels , Genetics , Metabolism , Evolution, Molecular , GRB7 Adaptor Protein , Chemistry , Genetics , Metabolism , HEK293 Cells , Light , Models, Molecular , Molecular Sequence Data , Protein Binding , Radiation Effects , Protein Structure, Tertiary , Protein Transport , Rod Cell Outer Segment , Radiation Effects , rab5 GTP-Binding Proteins , Metabolism , ras Proteins , MetabolismABSTRACT
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.
Subject(s)
Cattle , Rats , Aging , Animals , Brain/metabolism , Calcium/pharmacology , Comparative Study , GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Molecular Weight , Phosphorylation/drug effects , Rats, Sprague-Dawley , Synaptic Membranes/metabolism , Synaptosomes/metabolism , cdc42 GTP-Binding Protein/biosynthesis , rab3A GTP-Binding Protein/metabolism , rab5 GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/biosynthesisABSTRACT
Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions, i.e. the regulation of cytoskeletal organization in response to extracelluar growth factors and in dendritic neuron development. In this study, we have examined the regulation of small GTP-binding proteins that are implicated in neurosecretion and differentiation of neuron during ageing processes. Comparison of small GTP-binding proteins from the synaptosome and crude synaptic vesicles (LP2 membranes) of 2 months and 20 months old rat brain respectively showed no difference in the level of Rab family proteins (Rab3A and Rab5A). However, Rho family proteins such as RhoA and Cdc42 were elevated in LP2 membranes of the aged brain. The dissociation of Rab3A by Ca2+/calmodulin (CaM) from SV membranes was not changed during aging. Ca2+/CaM stimulated phosphorylation of the 22 and 55-kDa proteins in SV membranes from the aged rat brain, and inhibited phosporylation of 30-kDa proteins. GTPgammaS inhibited phosphorylation of the 100-kDa proteins and stimulated phosphorylation of the 70 kDa in LP2 membranes from both the young and aged rat brains, whereas GDPbetaS caused just the opposite reaction. These results suggest that protein phosphorylation and regulation of Rho family GTPases in rat brain appears to be altered during ageing processes.