We have previously characterized the release of the signal peptide sequence-less fibroblast growth factor (FGF) prototype, FGF-1, in vitro as a stress-induced pathway in which FGF-1 is released as a latent homodimer with the p40 extravesicular domain of p65 synaptotagmin (Syn)-1. To determine the biologic relevance of the FGF-1 release pathway in vivo, we sought to resolve and characterize from ovine brain a purified fraction that contained both FGF-1 and p40 Syn-1 and report that the brain-derived FGF-1:p40 Syn-1 aggregate is associated with the calcium-binding protein, S100A13. Since S100A13 binds the anti-inflammatory compound amlexanox and FGF-1 is involved in inflammation, we examined the effects of amlexanox on the release of FGF-1 and p40 Syn-1 in response to stress in vitro. We report that while amlexanox was able to repress the heat shock-induced release of FGF-1 and p40 Syn-1 in a concentration-dependent manner, it had no effect on the constitutive release of p40 Syn-1 from p40 Syn-1 NIH 3T3 cell transfectants. These data suggest the following: (i) FGF-1 is associated with Syn-1 and S100A13 in vivo; (ii) S100A13 may be involved in the regulation of FGF-1 and p40 Syn-1 release in response to temperature stress in vitro; and (iii) the FGF-1 release pathway may be accessible to pharmacologic regulation.
RNA polymerase II (Pol II), whose 12 subunits are conserved across eukaryotes, is at the heart of the machinery responsible for transcription of mRNA. Although associated general transcription factors impart promoter specificity, responsiveness to gene-and tissue-selective activators additionally depends on the multiprotein Mediator coactivator complex. We have isolated from tissue extracts a distinct and abundant mammalian Pol II subpopulation that contains an additional tightly associated polypeptide, Gdown1. Our results establish that Gdown1-containing Pol II, designated Pol II(G), is selectively dependent on and responsive to Mediator. Thus, in an in vitro assay with general transcription factors, Pol II lacking Gdown1 displays unfettered levels of activator-dependent transcription in the presence or absence of Mediator. In contrast, Pol II(G) is dramatically less efficient in responding to activators in the absence of Mediator yet is highly and efficiently responsive to activators in the presence of Mediator. Our results reveal a transcriptional control mechanism in which Mediatordependent regulation is enforced by means of Gdown1, which likely restricts Pol II function only to be reversed by Mediator.activator ͉ transcription ͉ repression ͉ negative regulator
Objective LRP1 is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells (SMC). Mice in which the lrp1 gene is deleted in SMC (smLRP1-/-) on an LDLr-deficient background display excessive PDGF signaling, SMC proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current studies were to examine the potential of LRP1 to modulate vascular physiology under non-atherogenic conditions. Approach and Results We found smLRP1-/- mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not due to excessive PDGF signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1-/- vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1) which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis resulting in fragmentation of elastic fibers. Importantly, our studies discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor (CTGF), an LRP1 ligand and a key mediator of fibrosis. Conclusions Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and CTGF protein levels. These studies highlight two new molecules, CTGF and HtrA1, which contribute to detrimental changes in the vasculature and therefore represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.
Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor.
Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.
The contribution of individual basic amino acids within three putative "consensus sequences" for heparin binding of fibroblast growth factor-1 have been examined by site-directed mutagenesis. The results indicate that a significant reduction in the apparent affinity of fibroblast growth factor-1 for heparin is only observed when basic residues in one of the three regions are mutated. Mutation in the other regions are without affect on heparin binding. The heparin binding properties of synthetic peptides based on the three "consensus sequences" paralleled the mutagenesis results. That is, synthetic peptides corresponding to regions of the protein that were affected by mutagenesis with respect to heparin binding exhibited a relatively high affinity for immobilized heparin, whereas those corresponding to regions of similar charge density that were unaffected by mutagenesis did not. In addition, amino acid substitution of a nonbasic residue in the heparin-binding peptide could abolish its heparin binding capacity. The heparin-binding peptide could antagonize the mitogenic activity of FGF-1, probably because of the heparin dependence of this activity. Together these data demonstrate that the heparin binding properties of fibroblast growth factor-1 are dictated by structural features more complex than clusters of basic amino acids. The results of these and other studies indicate that consensus motifs for heparin-binding require further definition. More importantly, the results provide a basis for the design of peptide-based inhibitors of FGF-1.The fibroblast growth factor (FGF) 1 family consists of at least nine structurally related proteins (1-3). Two of these proteins, FGF-1 and FGF-2, have been characterized under many different names, most often as acidic FGF and basic FGF, respectively. Although there is a large amount of overlap in the spectrum of biological activities and receptor-binding properties of the FGFs, the only known function shared by all members of the family is a relatively high affinity for heparin or heparan sulfate proteoglycans (HSPGs). It has been established that heparin can potentiate the mitogenic activity of FGF-1 (4 -6) and protect both FGF-1 and FGF-2 from proteolytic and heat inactivation (7-9). In addition, heparin increases the apparent affinity of FGF-1 for high affinity FGF receptors (10, 11). Recently, an obligatory role for heparin or HSPGs in mediating the binding of FGF-1 or FGF-2 to the high affinity, tyrosine kinase receptors has been suggested (12-17). It has also been reported that cell surface HSPGs are capable of binding and internalizing FGF-2 (18). A direct role of HSPGbound FGF in mediating the various functions of this growth factor family has not been established.We reported previously that a change of lysine 132 in FGF-1 to a glutamic acid (K132E) by site-directed mutagenesis reduced the apparent affinity of the recombinant protein for heparin (4). The K132E mutant is fully capable of binding to and activating the high-affinity tyrosine kinase FGF receptors and can i...
The heparin-binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF activities. In this report, we summarize evidence that indicates that the heparin-binding and mitogenic activities of HBGF-1 can be dissociated from the receptor-binding activities of the growth factor by site-directed mutagenesis of a single lysine residue. Thus, the mutant HBGF-1 has normal receptor-binding activity and is capable of stimulating tyrosine kinase activity and proto-oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto-oncogene expression from the mitogenic response is observed when the human wild-tupe HBGF-1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild-type or mutant HBGF-1. Production of wild-type induces a transformed phenotype, whereas over-production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site-directed mutagenesis of putative nuclear translocation sequences in the wild-type protein do not affect mitogenic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the alpha catalytic subunit of protein kinase CK2 (CK2alpha) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2alpha and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.
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