IntroductionAdult stem cells and progenitor cells can integrate and respond to appropriate extracellular stimuli in the form of hormones and growth factors, or contact with the extracellular matrix (ECM) and neighboring cells. The delicate balance between the dormancy of progenitor cells and their timely proliferation and differentiation is a crucial parameter in tissue homeostasis that is often perturbed in disease. In vitro studies and animal implant experiments have revealed the multipotential nature of mesenchymal stem cells, which contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, tendon, stroma and adipose tissues (Pittenger et al., 1999;Prockop, 1997).Adipogenesis is a complex process characterized by the strict temporal regulation of multiple and interacting signaling events that ultimately lead to the expression of adipocytespecific genes (Gregoire et al., 1998;Rosen and Spiegelman, 2000). A cascade of transcription factors is induced that involves the sequential activation of the CCAAT/enhancerbinding proteins (C/EBPs) and the peroxisome proliferatoractivated receptor γ (PPARγ), which leads to the activation of several genes, such as those responsible for lipid transport and metabolism. Initially, fibroblastic preadipocytes stop dividing and acquire a rounded morphology. The change of shape from fibroblastic preadipocytes to rounded, mature adipocytes is accompanied by changes in cytoskeletal organization and contacts with the ECM. The expression of fibronectin, integrins, actin and several cytoskeletal proteins is downregulated during adipogenesis (Rodriguez Fernandez and Ben-Ze'ev, 1989;Spiegelman and Farmer, 1982). In fact, the disruption of contacts with the ECM is a requirement for adipocyte differentiation (Spiegelman and Ginty, 1983).Our understanding of the molecular events involved in adipogenesis is based mainly on in vitro, cell culture models, 3893Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of β1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/β1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal ad...
IntroductionFms-like tyrosine kinase 3 (Flt3) functions as a growth factor receptor and is expressed primarily in multipotential hematopoietic stem cells and progenitors as well as in placenta, gonads, and brain. Together with its activating ligand Flt3 ligand (FL) it is a crucial player in assuring normal function of stem cells and the immune system. [1][2][3] Moreover, approximately 30% to 35% of patients with acute myeloid leukemia (AML) carry a mutation in Flt3, rendering Flt3 the most frequently mutated gene in AML. 4,5 Flt3 mutations are generally grouped into 2 classes: point mutations in the vicinity of codon 835 or 842 within the tyrosine kinase domain (TKD); or internal tandem duplications (ITDs) of varying lengths within the juxtamembrane domain of Flt3, which sterically represses the intrinsic kinase activity of Flt3 in the absence of ligand. 4,6 Both classes of mutations result in constitutive activation of Flt3 but distinct signaling and transforming capacities. 7-10 Although debatable as prognostic markers by themselves, ITD and TKD mutations are correlated with poor prognostic features for AML patients, suggesting Flt3 or one of its downstream effectors as potential therapeutic targets. [11][12][13][14][15] Flt3 constitutes, together with the receptor for stem cell factor (c-Kit), the receptors for platelet-derived growth factors (PDGFRs), and colony-stimulating factor-1 (CSF-1), the type III family of receptor tyrosine kinases (RTKs). 5 Type III RTKs share a common modular structure consisting of 5 extracellular Ig-like domains, a short transmembrane stretch, a juxtamembrane region followed by a bipartite kinase domain interrupted by the kinase insert, and the carboxyterminal tail. 16 Ligand binding causes receptor dimerization, kinase activation, and transphosphorylation of RTK on multiple tyrosine residues. 17 These autophosphorylated tyrosine residues together with 3 to 6 adjacent amino acids form high-affinity docking sites for relay molecules possessing either phosphotyrosine binding (PTB) or Src homology 2 (SH2) domains. 18 Upon relocation to the receptor, these signaling or adapter molecules become activated in either a phosphorylation-dependent or -independent manner and are thereby capable of transducing the signal downstream. Relay molecules reported to be recruited and/or activated upon Flt3 activation include the p85 subunit of PI3K, Ras-GAP, PLC-␥, Vav, SHIP1, SHP2, ShcA, Grb2, Cbl, and Src family kinases (SFKs) as well as Stat5. [19][20][21][22][23][24][25][26] Whereas the immediate signaling steps following ligand binding (ie, binding of signaling molecules to autophosphorylated tyrosines) are well studied in the c-Kit, PDGFRs, and CSF-1 receptor systems, 16 For personal use only. on June 19, 2019. by guest www.bloodjournal.org From Y589, Y591, Y597, or Y599, which could theoretically add to the aberrant signal relay from the autoactivated receptor. 27 Here we report that Y572, Y589, Y591, and Y599 of Flt3 are phosphorylated in vivo in Flt3-expressing cells following ligand stimu...
ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane-anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did not exhibit increased serum insulin, cholesterol, or triglycerides. Male transgenics were slightly overweight and also developed herniation but did not become obese. Transgenic mice expressing a truncated form of ADAM 12-S lacking the prodomain and the metalloprotease domain did not develop this adipogenic phenotype, suggesting a requirement for ADAM 12 protease activity. This is the first in vivo demonstration that an ADAM protease is involved in adipogenesis.
The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembraneanchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) ⑀ induces ADAM12 translocation to the cell surface and that catalytic activity of PKC⑀ is required for this translocation. Cells possess a diverse array of surface proteins, lipids, and carbohydrates that provide active gateways for the selective intake and release of molecular information, which is important in regulating cell behavior. In fact, many disease processes relate to disorganized cell-surface communication systems. ADAMs 1 belong to a large family of cell-surface proteins with over 30 members. The prototypical ADAM molecule is a transmembrane glycoprotein composed of several distinct domains, including a prodomain and a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, transmembrane, and cytoplasmic domain. ADAMs play important roles in cell adhesion, interacting with integrins and syndecans, and in the proteolysis of the ectodomains of cell-surface proteins,
Platelet-derived growth factor-stimulated actin rearrangement and edge ruffle formation have previously been shown to be dependent on activation of phosphatidylinositol 3′-kinase, the activity of which also is important for directed migration of cells. This lipid kinase binds to phosphorylated tyrosine residues Y740 and Y751 in the kinase insert of the human platelet-derived growth factor ss-receptor. We examined the role of two other tyrosine residues in the kinase insert of this receptor, Y775 and Y778, for ligand-induced actin rearrangement. Both were shown to be phosphorylation sites; Y775 was only marginally phosphorylated in cells expressing the wild-type ss-receptor, whereas Y778 was phosphorylated at higher stoichiometry. Mutant receptors Y775F, Y778F and Y775/778F were active kinases and mediated proliferative responses when expressed in porcine aortic endothelial cells. Fluorescence staining of actin in platelet-derived growth factor-stimulated PAE cells revealed that Y778 is involved in regulation of the actin cytoskeleton since the cells contained, apart from edge ruffles and circular ruffles, a novel type of giant ruffle on the dorsal side of the cell, which consisted of irregular multilayered actin structures. Mutation at Y778 had no effect on activation of phosphatidylinositol 3′-kinase, nor on the GTPase activating protein of Ras and phospholipase C(gamma), and the extent of directed migration towards platelet-derived growth factor of these cells was not changed. We conclude that actin rearrangement is regulated in part by Y778 in the platelet-derived growth factor ss-receptor, potentially through binding of a novel signaling molecule to this site.
Early signal relay steps upon ligand-binding to the receptor tyrosine kinase Flt3, i.e. sites of Flt3-autophosphorylation and subsequent docking partners, are mainly unresolved. Here we demonstrate for the first time identification of ligand-induced in vivo phosphorylation sites in Flt3. By immunoprecipitation of specific tryptic peptides contained in the juxtamembrane region of human Flt3 and subsequent radiosequencing we identified the tyrosine residues 572, 589, 591 and 599 as in vivo autophosphorylation sites. Focusing on Y589 and Y599, we examined Flt3-ligand-mediated responses in WT-Flt3, Y589F-Flt3 and Y599F-Flt3 expressing 32D cells. Compared to WT-Flt3-32D cells, 32D-Y589F-Flt3 showed upon ligand-stimulation enhanced Erk activation as well as proliferation/survival whereas 32D-Y599F-Flt3 cells displayed substantially diminished responses. Both pY589 and pY599 were identified as association sites for multiple signal relay molecules including Src family kinases. Consistently, 32D-Y589F-Flt3 and 32D-Y599F-Flt3 showed decreased FL-triggered Src activation, impaired phosphorylation of the adapter molecules Cbl and ShcA and deficient receptor ubiquitination and degradation. Interference with the Src-dependent negative regulation of Flt3 signaling may account for the enhanced mitogenic response of Y589F-Flt3. pY599 was additionally found to interact with the protein tyrosine phosphatase Shp2. As Y599F-Flt3-32D lacked ligand-induced Shp2 phosphorylation and since silencing of Shp2 in WT-Flt3-expressing cells mimicked the Y599F-Flt3-phenotype we hypothesize that recruitment of Shp2 to pY599 contributes to FL-mediated Erk activation and proliferation. To summarize, our work presents novel insights in Flt3-mediated signal transduction. We have identified the in vivo autophosphorylation sites of the juxtamembrane region of Flt3, revealed Src family kinases and Shp2 as binding partners of pY589 and/or pY599, respectively, as well as their potential impact on FL-mediated signaling in Flt3-32D cells. Future work will now focus on elucidation of additional and possibly novel interaction partners of the found phosphorylation sites by employing an unbiased proteomics approach. With this gained knowledge it will be of interest to see whether ITDs differing in the nature of the duplicated tyrosines also confer distinct signaling behavior. If so, these tyrosines might serve as a diagnostic marker and point towards a successful combinatorial therapy consisting of a receptor tyrosine kinase inhibitor and an inhibitor for the specifically affected signal transduction pathway.
c-Kit is a member of the receptor tyrosine kinase family class III. Upon stimulation with its ligand, stem cell factor, c-Kit becomes activated and phosphorylated on tyrosine residues. The receptor activation leads to cellular responses such as proliferation, differentiation, survival and migration. c-Kit is known to be involved in the pathogenesis of several forms of cancer, e.g. gastrointestinal stroma tumors, leukemias and small-cell lung cancer. c-Kit exists in different isoforms due to alternative mRNA splicing. We are studying one pair of isoforms which varies by the presence or absence of four amino acids GNNK, in the juxtamembrane region just outside the plasma membrane. They are denoted GNNK− and GNNK+, respectively, and have previously been shown by our group to display functional differences in the activation of signaling pathways and cellular responses in the fibroblast cell line NIH3T3. The GNNK− splice form binds and activates Src family kinases more efficiently than the GNNK+ splice form. This leads to stronger phosphorylation of ShcA, extracellular signal regulated kinase 1/2 (Erk) and Cbl compared to the GNNK+ form. In order to study the two GNNK spliceforms in a more physiological context, we have retrovirally transduced two hematopoietic cell lines, 32D and BaF/3, which we now use as our model system. We could demonstrate that SCF stimulation induced a kinetically faster and stronger tyrosine phosphorylation pattern in the GNNK− splice form than the c-Kit GNNK+ splice form. Moreover, the phosphorylation of Erk and Shc is stronger in the c-Kit GNNK− splice form. These results are in line with the results described for NIH3T3 fibroblasts and previously published by our group. We are investigating the SCF-induced proliferative response in the hematopoietic cell lines 32D and BaF/3. Having established these model cell lines, we now aim to use Affymetrix microarray technology to detect differences in gene expression between the two splice forms upon ligand stimulation for different periods of time. As these different splice forms show remarkable differences in their signaling capacity we expect to find significant differences, but also similarities, in gene induction/repression mediated by the c-Kit splice forms. The idea is to link these findings to the biological responses induced by the two splice forms. We will more thoroughly investigate these findings using different c-Kit mutants and a battery of inhibitors of signaling proteins of interest available in the lab. An increased knowledge of c-Kit signaling in general and of the two different c-Kit splice forms in particular will help to find rational treatment for the unfavorable effect of c-Kit in cancer.
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