Using a mRNA differential screening of fibroblasts differing for the expression of c-fos we isolated a c-fos-induced growth factor (FIGF). The deduced protein sequence predicts that the cDNA codes for a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. Northern blot analysis shows that FIGF expression is strongly reduced in c-fos-deficient cells. Transfection of exogenous c-fos driven by a constitutive promoter restores the FIGF expression in these cells. In contrast, both PDGF and VEGF expression is unaffected by c-fos. FIGF is a secreted dimeric protein able to stimulate mitogenic activity in fibroblasts. FIGF overexpression induces morphological alterations in fibroblasts. The cells acquire a spindle-shaped morphology, become more refractive, disorganized, and detach from the plate. These results imply that FIGF is a downstream growth and morphogenic effector of c-fos. These results also suggest that the expression of FIGF in response to c-fos activation induces specific differentiation patterns and its aberrant activation contributes to the malignant phenotype of tumors.

Protein kinase CK2 is an ubiquitous and pleiotropic Ser/Thr protein kinase composed of two catalytic (␣ and/or ␣) and two noncatalytic (␤) subunits forming a heterotetrameric holoenzyme involved in cell growth and differentiation. Here we report the identification, cloning, and oncogenic activity of the murine CK2␣ subunit. Serum treatment of quiescent mouse fibroblasts induces CK2␣ mRNA expression, which peaks at 4 h. The kinetics of CK2␣ expression correlate with increased kinase activity toward a specific CK2 holoenzyme peptide substrate. The ectopic expression of CK2␣ (or CK2␣) cooperates with Ha-ras in foci formation of rat primary embryo fibroblasts. Moreover, we observed that BALB/c 3T3 fibroblasts transformed with Ha-ras and CK2␣ show a faster growth rate than cells transformed with Ha-ras alone. In these cells the higher growth rate correlates with an increase in calmodulin phosphorylation, a protein substrate specifically affected by isolated CK2 catalytic subunits but not by CK2 holoenzyme, suggesting that unbalanced expression of a CK2 catalytic subunit synergizes with Ha-ras in cell transformation.Protein kinase CK2 (previously known as casein kinase II) is an ubiquitous Ser/Thr kinase present in the cytoplasm and the nucleus of eukaryotic cells (for review, see Refs. 1-5). CK2 holoenzyme consists of two catalytic (␣ and/or ␣Ј) and two regulatory (␤) subunits assembled as stable heterotetramers, which in vitro do not dissociate unless under denaturing conditions. CK2 is unique among Ser/Thr protein kinases for its ability to use GTP, besides ATP, as phosphate donor and for its unusual site specificity, which is determined by multiple acidic and/or previously phosphorylated residues downstream (nϩ3) from the phosphoacceptor amino acid, determining the minimum consensus (S/T-X-X-E/D/Yp/Sp) (6).More than 160 cellular proteins have been reported to be phosphorylated by CK2, and several are implicated in signal transduction, transcriptional activation, cell cycle progression, and cell differentiation. The nuclear proteins that are CK2 substrates includes: c-Myc (7), Max (7), c-Myb (8), serum response factor (SRF) (9), DNA ligase I (10), DNA topoisomerase 2 (11), p53 (12), and c-Fos (13). In mammalian cells phosphorylation of nuclear factors dependent on CK2 could be relevant for cell growth regulation and the progression into the cell cycle. A direct role of CK2 activity in cell cycle progression has been demonstrated by antibody-mediated CK2 depletion and by gene inactivation in Saccharomyces cerevisiae (14, 15). Although hundreds of papers have been published on the subject, it is still unknown how the enzyme is regulated in vivo (4, 5, 16). CK2␤ undergoes stoichiometric autophosphorylation and both CK2␤ and CK2␣ (but not CK2␣Ј) are phosphorylated in vitro and in vivo by p34Cdc2 kinase (17). However, these phosphorylations do not correlate with any regulation of activity. Moreover, it is not clear whether the holoenzyme represents an up-or a down-regulated form of the kinase, because some substrates ar...

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

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Key Words: cell adhesion Ⅲ endothelial cells Ⅲ angiogenesis Ⅲ biochemistry Ⅲ signaling C rosstalk between integrins and growth factor receptors plays an important role in vascular development and its maintenance. Several examples demonstrated that crosstalk between integrins and tyrosine kinase receptors is required for growth factor-induced biological processes to ensure cell growth, survival, and differentiation in normal and pathological processes. 1 The angiogenic processes require the coordination of signals from the extracellular environment to activates specific tyrosine kinase receptors and integrins. 1,2 Integrins associating with growth factor receptors regulate the capacity of the integrin/receptor complexes to propagate downstream signals. 3,4 Integrin-dependent activation of receptor tyrosine kinases is a general mechanism to enhance growth factor signals, the recruitment of transducing proteins to membrane cytoskeletal complexes as well as nuclear responses. This cooperation has been shown for several signaling pathways including insulin, epidermal growth factor (EGF), platelet-derived growth factor, fibroblast growth factor, and vascular endothelial growth factor (VEGF) signaling. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] Integrins are able to trigger ligand-independent EGF receptor autophosphorylation leading to activation of the downstream pathway. 16 Integrins, c-Src, p130Cas and EGF receptor associate in a macromolecular complex on the cell membrane and integrin-dependent adhesion induces phosphorylation of specific tyrosine residues of EGF receptor, distinct from those obtained by the soluble ligand EGF. 11,19 A large body of evidence demonstrates that the angiogenic process is initiated by mitogenic signals induced by growth factors such as VEGF and the interplay between the tyrosine 34 -36 VEGFR-3, in contrast to its highly related endothelial receptor VEGFR-2 (which, after VEGF stimulation, forms a complex with integrin ␤3 subunit 8,20,21,24,25,37 ), associates selectively with integrin ␤1. 10,12 Cell attachment to fibronectin or collagen induces the phosphorylation of VEGFR-3 in the absence of a ligand and significantly enhances the phosphorylation of the receptor induced by its ligand. 10,12 However, the mechanism of integrin activation of VEGFR-3 has not been clarified.Here, we demonstrate that collagen I-induced activation of VEGFR-3 is independent from the intrinsic catalytic activity of the receptor but it is a direct target of c-Src. c-Src phosphorylates VEGFR-3 at specific tyrosine residues with a pattern of phosphorylation that is distinct from the pattern induced by the ligand. Cell adhesion induces the phosphorylation of the tyrosine residues 1063 and 1337, which are known binding sites for CRKI/II and SHC. Pull-down assays demonstrated that integrin-mediated receptor phosphorylation induces the recruitment of CRKI/II and SHC second messengers to the receptor suggesting that the integrin/receptor complex can activate growth and survival signaling in the absence ...