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PubMed2001

Hepatocyte growth factor induction of collagenase 3 production in human osteoarthritic cartilage: involvement of the stress-activated protein kinase/c-Jun N-terminal kinase pathway and a sensitive p38 mitogen-activated protein kinase inhibitor cascade.

OBJECTIVE: Osteoarthritis (OA) involves both a decreased reparative process and an increased degradative phenomenon. Several cytokines and growth factors are known to facilitate the repair of articular cartilage defects. The hepatocyte growth factor (HGF) present in OA cartilage is suggested to be involved in the cartilage repair process as well as in matrix remodeling and chondrocyte migration, leading to partial reconstruction of articular cartilage. Since cell migration is often correlated with metalloprotease activity, the effect of HGF on collagenase 3 production was studied because of its possible implication in OA cartilage remodeling. METHODS: We examined HGF-stimulated collagenase 3 production in human OA chondrocytes by Western and Northern blotting. Furthermore, we explored the intracellular signaling pathways through which HGF induced collagenase 3 production. RESULTS: This study showed that HGF stimulated collagenase 3 production in human OA chondrocytes at the transcriptional level, and this induction was mediated by activation of the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway, but not the p38 mitogen-activated protein kinase (MAPK). The p44/42 MAPKs were also phosphorylated and the use of their specific inhibitor (PD 98059) did not affect HGF-induced collagenase 3 production in OA chondrocytes. Induced collagenase 3 production via the SAPK/JNK pathway was mediated, at least in part, by the TRE site in the promoter, and in the activator protein 1 complex, c-Jun, JunD, and Fra-1 were activated. Surprisingly, further experiments revealed that the specific p38 MAPK inhibitor SB 202190 also inhibited collagenase 3 production early in the HGF-induced process. The 50% inhibitory concentration was as low as 50 nM, which is unlikely to be related to p38 MAPK inhibition (which is usually in the microM range), suggesting the involvement of another kinase sensitive to SB 202190. CONCLUSION: This is the first study to show that HGF has the ability to induce both the expression and synthesis of collagenase 3 in OA chondrocytes. The effect is mediated by kinase cascades involving SAPK/JNK and another, unidentified kinase. This study provides novel information implicating a role for HGF in the pathophysiology of OA through its effect on the production of collagenase 3, which is an enzyme that is possibly involved in OA cartilage remodeling.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The postsynaptic scaffold proteins ProSAP1/Shank2 and Homer1 are associated with glutamate receptor complexes at rat retinal synapses.

The postsynaptic density (PSD) at glutamatergic synapses is a macromolecular complex of various molecules that organize the different glutamate receptors spatially and link them to their appropriate downstream signaling pathways and to the cytoskeleton. Recently, a new family of multidomain proteins called Shanks or ProSAPs (proline-rich synapse-associated proteins) has been identified. They are suggested to be central adaptor proteins of the PSD of glutamatergic synapses, bridging different types of glutamate receptor complexes. With immunocytochemistry and light and electron microscopy, we examined the cellular, synaptic, and postnatal developmental expression of ProSAP1/Shank2 at the synapses of rat retina. With double-labeling experiments and confocal microscopy, we analyzed the association of ProSAP1/Shank2 with proteins specific for glutamatergic, glycinergic, and gamma-aminobutyric acid (GABA)ergic synapses and with proteins known to be involved in the structural and functional organization of PSDs containing N-methyl-D-aspartate receptors [95-kDa postsynaptic density protein (PSD-95)], group I metabotropic glutamate receptors (Homer1), and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors [glutamate receptor-interacting protein (GRIP)]. ProSAP1/Shank2 was present postsynaptically at the glutamatergic ribbon synapses of photoreceptor and bipolar cells, and it was absent from glycinergic and GABAergic amacrine cell synapses. The double-labeling experiments revealed a high rate of colocalization of ProSAP1/Shank2 with Homer1 and PSD-95, and little colocalization with GRIP. These data suggest that ProSAP1/Shank2 acts as an organizer at PSDs of different glutamatergic retinal synapses.

Animals↗
PubMed2005

ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity.

Apoptosis signal-regulating kinase 1 (ASK1) is an evolutionarily conserved mitogen-activated protein 3-kinase that activates both Jnk and p38 mitogen-activated protein kinases. Here we used ASK1-deficient mice to show that ASK1 was selectively required for lipopolysaccharide-induced activation of p38 but not of Jnk or the transcription factor NF-kappaB. ASK1 was required for the induction of proinflammatory cytokines dependent on Toll-like receptor 4 (TLR4) but not TLR2 or other TLRs. Consistent with this, ASK1-deficient mice were resistant to lipopolysaccharide-induced septic shock. Lipopolysaccharide induced the production of intracellular reactive oxygen species, which was required for the formation of a complex of the adaptor molecule TRAF6 and ASK1 and subsequent activation of the ASK1-p38 pathway. Our data demonstrate that the reactive oxygen species-dependent TRAF6-ASK1-p38 axis is crucial for TLR4-mediated mammalian innate immunity.

Animals↗
PubMed2003

Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkappa B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR .

Recent studies show that a member of the interleukin-1 (IL-1)/Toll receptor superfamily, Toll-like receptor 3 (TLR3), recognizes double-stranded RNA (dsRNA). Because of the similarity in their cytoplasmic domains, IL-1/Toll receptors share signaling components that associate with the IL-1 receptor, including IL-1 receptor-associated kinase (IRAK), MyD88, and TRAF6. However, we find that, in response to dsRNA, TLR3 can mediate the activation of both NFkappaB and mitogen-activated protein (MAP) kinases in IL-1-unresponsive mutant cell lines, including IRAK-deficient I1A and I3A cells, which are defective in a component that is downstream of IL-1R but upstream of IRAK. These results clearly indicate that TLR3 does not simply share the signaling components employed by the IL-1 receptor. Through biochemical analyses we have identified an IRAK-independent TLR3-mediated pathway. Upon binding of dsRNA to TLR3, TRAF6, TAK1, and TAB2 are recruited to the receptor to form a complex, which then translocates to the cytosol where TAK1 is phosphorylated and activated. The dsRNA-dependent protein kinase (PKR) is also detected in this signal-induced TAK1 complex. Kinase inactive mutants of TAK1 (TAK1DN) and PKR (PKRDN) inhibit poly(dI.dC)-induced TLR3-mediated NFkappaB activation, suggesting that both of these kinases play important roles in this pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Analysis of tyrosine phosphorylation-dependent interactions between stimulatory effector proteins and the B cell co-receptor CD22.

The B cell-restricted transmembrane glycoprotein CD22 is rapidly phosphorylated on tyrosine in response to cross-linking of the B cell antigen receptor, thereby generating phosphotyrosine motifs in the cytoplasmic domain which recruit intracellular effector proteins that contain Src homology 2 domains. By virtue of its interaction with these effector proteins CD22 modulates signal transduction through the B cell antigen receptor. To define further the molecular mechanism by which CD22 mediates its co-receptor function, phosphopeptide mapping experiments were conducted to determine which of the six tyrosine residues in the cytoplasmic domain are involved in recruitment of the stimulatory effector proteins phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI3K), Grb2, and Syk. The results obtained indicate that the protein tyrosine kinase Syk interacts with multiple CD22-derived phosphopeptides in both immunoprecipitation and reverse Far Western assays. In contrast, the Grb2.Sos complex was observed to bind exclusively to the fourth phosphotyrosine motif (Y828ENV) from CD22 and does so via a direct interaction based on Far Western and reverse Far Western blotting. Although both PLCgamma and PI3K were observed to bind to multiple phosphopeptides in precipitation experiments, subsequent studies using reverse Far Western blot analysis demonstrated that only the carboxyl-terminal phosphopeptide of CD22 (Y863VTL) binds directly to either one. This finding suggests that PLCgamma and PI3K may be recruited to CD22 either through a direct interaction with Tyr863 or indirectly through an association with one or more intermediate proteins.

Adaptor Proteins, Signal Transducing↗
PubMed2003

SETA/CIN85/Ruk and its binding partner AIP1 associate with diverse cytoskeletal elements, including FAKs, and modulate cell adhesion.

The adaptor protein SETA/CIN85/Ruk is involved in regulating diverse signal transduction pathways, including the internalization of tyrosine kinase receptors via the Cbl ubiquitin ligases, and attenuating PI3K activity by interaction with its regulatory subunit. Here we present evidence for a new aspect of SETA function, based on the initial observation that it co-localizes with actin in microfilaments and at focal adhesions, and with microtubules. Although there was no evidence for direct molecular interactions between SETA and cytoskeletal proteins, the SETA-interacting protein AIP1, which is a rat ortholog of the Xenopus src substrate Xp95, strongly interacted with structural proteins of the cytoskeleton, including actin and tubulins. Both SETA and AIP1 interacted with focal adhesion kinase (FAK) and proline rich tyrosine kinase 2 (PYK-2), and c-Cbl interacted with PYK-2. AIP1, which interacted more strongly than either SETA or c-Cbl, required an intact consensus tyrosine kinase phosphorylation sequence at Y319 to bind to focal adhesion kinases, which suggests that phosphorylation is an important mediator of this complex. SETA, which interacted as a dimer with focal adhesion kinases, promoted the interaction between PYK-2 and AIP1. Direct analysis of the impact of these proteins on cell adhesion, by use of an electrical cell-substrate impedance sensor (ECIS), showed that SETA promoted cell adhesion while AIP1 and c-Cbl reduced it. Furthermore, the ability of AIP1 and AIP1 mutants to decrease cell adhesion in ECIS analysis correlated with their presence in PYK-2 complexes, providing a direct link between AIP1-mediated molecular interactions and cellular behavior. Transfection of AIP1 also reduced the level of phosphorylation of endogenous PYK-2 and FAK, suggesting that this protein may directly regulate focal adhesion kinases, and thereby cell adhesion. These data are the first to implicate the adaptor protein SETA and its binding partner AIP1 as being involved with the cytoskeleton and in the regulation of cell adhesion, and suggest that they may be part of the focal adhesion kinase regulatory complex.

Actins↗
PubMed2006

Rapamycin activates Tap42-associated phosphatases by abrogating their association with Tor complex 1.

In Saccharomyces cerevisiae, the Tap42-phosphatase complexes are major targets of the Tor kinases in the rapamycin-sensitive signaling pathway. The immunosuppressive agent, rapamycin, induces a prompt activation of the Tap42-associated phosphatases, which is vitally important in Tor-mediated transcriptional regulation. However, the mechanism for the rapid phosphatase activation is poorly understood. In this study, we show that the Tap42-phosphatase complexes exist mainly on membrane structures through their association with Tor complex 1 (TORC1). Rapamycin abrogates this association and releases the Tap42-phosphatase complexes into the cytosol. Disassembly of the Tap42-phosphatase complexes occurs subsequently, following the release but at a much slower rate, presumably caused by Tap42 dephosphorylation. Release of the Tap42-phosphatase complexes from membrane structures also occurs when cells are deprived of nutrient. These findings suggest that the association of the Tap42-phosphatase complexes with TORC1 represents an important mechanism by which nutrient controls Tor signaling activity. In addition, our data support a model in which rapamycin acts not by inhibiting the kinase activity of Tor but by disrupting its interaction with downstream targets.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Lack of acrosome formation in Hrb-deficient mice.

The sperm acrosome is essential for sperm-egg fusion and is often defective in men with nonobstructive infertility. Here we report that male mice with a null mutation in Hrb are infertile and display round-headed spermatozoa that lack an acrosome. In wild-type spermatids, Hrb is associated with the cytosolic surface of proacrosomic transport vesicles that fuse to create a single large acrosomic vesicle at step 3 of spermiogenesis. Although proacrosomic vesicles form in spermatids that lack Hrb, the vesicles are unable to fuse, blocking acrosome development at step 2. We conclude that Hrb is required for docking and/or fusion of proacrosomic vesicles during acrosome biogenesis.

Acrosome↗
PubMed1998

A tripartite protein complex with the potential to couple synaptic vesicle exocytosis to cell adhesion in brain.

We identify a complex of three proteins in brain that has the potential to couple synaptic vesicle exocytosis to neuronal cell adhesion. The three proteins are: (1) CASK, a protein related to MAGUKs (membrane-associated guanylate kinases); (2) Mint1, a putative vesicular trafficking protein; and (3) Veli1, -2, and -3, vertebrate homologs of C. elegans LIN-7. CASK, Mint1, and Velis form a tight, salt-resistant complex that can be readily isolated. CASK, Mint1, and Velis contain PDZ domains in addition to other modules. However, no PDZ domains are involved in complex formation, leaving them free to recruit cell adhesion molecules, receptors, and channels to the complex. We propose that the tripartite complex acts as a nucleation site for the assembly of proteins involved in synaptic vesicle exocytosis and synaptic junctions.

Adaptor Proteins, Signal Transducing↗
PubMed2004

TAP/NXF1, the primary mRNA export receptor, specifically interacts with a neuronal RNA-binding protein HuD.

Hu proteins are RNA-binding proteins that are implicated in the control of stabilization, nuclear export, and/or translation of specific mRNAs with AU-rich elements (AREs) in the 3'-untranslated region. Three neuron-specific Hu proteins (HuD, HuB, and HuC), but not a ubiquitously expressed Hu protein HuR, have an activity to induce neurite outgrowth when they are overexpressed in a rat neuronal cell line PC12. Here we show that TAP/NXF1, the primary export receptor for the bulk mRNA, is a specific binding partner for HuD. In vitro binding experiments using recombinant proteins revealed that the interaction between TAP and HuD is direct and that HuD can form a ternary complex together with both TAP and RNA. Interestingly, HuR does not interact with TAP. These results suggest that HuD acts as a novel adaptor protein to recruit TAP for efficient export of ARE-containing mRNAs in neuronal cells.

Animals↗
PubMed1999

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Growth retardation as well as spleen and thymus involution in latent TGF-beta binding protein (Ltbp)-3 null mice.

The latent TGF-beta binding protein (LTBP)-3 is an extracellular matrix (ECM) protein that binds the small latent complex (SLC) of TGF-beta. Disruption of the Ltbp-3 gene by homologous recombination in mice yields mutant animals that display multiple skeletal abnormalities. In addition, these mice have retarded growth. On an inbred 129 SvEv background, half of the Ltbp-3 mutant mice die between 3 and 4 weeks after birth. These mice show severe involution of the thymus and spleen and a sharp reduction in the numbers of CD4/CD8 double positive T-cells in the thymus. The thymus and spleen defect is caused by elevated corticosterone levels in the serum and can be reversed by injection of aminoglutethimide (AMG), an inhibitor of steroid synthesis. This result indicates that the thymus and spleen defect is a secondary defect due to high corticosterone levels probably induced by stress of unknown etiology.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel.

Sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD) is mediated by beta adrenergic receptor (betaAR) activation, which increases the slow outward potassium ion current (IKS). Mutations in two human I(KS) channel subunits, hKCNQ1 and hKCNE1, prolong APD and cause inherited cardiac arrhythmias known as LQTS (long QT syndrome). We show that betaAR modulation of I(KS) requires targeting of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA) and protein phosphatase 1 (PP1) to hKCNQ1 through the targeting protein yotiao. Yotiao binds to hKCNQ1 by a leucine zipper motif, which is disrupted by an LQTS mutation (hKCNQ1-G589D). Identification of the hKCNQ1 macromolecular complex provides a mechanism for SNS modulation of cardiac APD through IKS.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Recruitment of the protein-tyrosine phosphatase SHP-2 to the C-terminal tyrosine of the prolactin receptor and to the adaptor protein Gab2.

The protein-tyrosine phosphatase SHP-2 modulates signaling events through receptor tyrosine kinases and cytokine receptors including the receptor for prolactin (PRLR). Here we investigated mechanisms of SHP-2 recruitment within the PRLR signaling complex. Using SHP-2 and PRLR immunoprecipitation studies in 293 cells and in the mouse mammary epithelial cell line HC11, we found that SHP-2 co-immunoprecipitates with the PRLR and that the C-terminal tyrosine of the PRLR plays a regulatory role in both the tyrosine phosphorylation and the recruitment of SHP-2. Our results further indicate that SHP-2 association to the PRLR occurs via the C-terminal SH2 domain of the phosphatase. In addition, we determined that the newly identified adaptor protein Gab2, but not Gab1, is specifically tyrosine phosphorylated and is able to recruit SHP-2 and phosphatidyinositol 3-kinase in response to PRLR activation. Together, these studies suggest the presence of dual recruitment sites for SHP-2; the first is to the C-terminal tyrosine of the PRLR and the second is to the adaptor protein Gab2.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Src-dependent association of Cas and p85 phosphatidylinositol 3'-kinase in v-crk-transformed cells.

Cellular changes associated with oncogenic transformation are generally caused by deregulation of signal transduction pathways. We show that, in cells transformed by the v-crk oncogene, the adapter protein Cas forms a stable complex with the p85 regulatory subunit of phosphatidylinositol 3'-kinase (PI3K) coincident with the appearance of Cas-associated PI3K activity. The interaction between Cas and p85 PI3K appears to be driven primarily by Src-dependent tyrosine phosphorylation of Cas, and mapping studies indicate that the carboxyl terminus of Cas is necessary and sufficient for binding to p85 PI3K. One of the cellular effects of v-Crk expression is to promote DNA synthesis in the presence of low serum. This effect is potentiated in Cas-null fibroblasts when wild-type Cas is expressed, but not when a Cas variant is expressed that lacks the carboxyl-terminal p85 PI3K binding region. This suggests that the association of Cas with p85 PI3K may play a role in uncoupling growth regulatory pathways through v-Crk.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

Growth factors induce differential phosphorylation profiles of the Hrs-STAM complex: a common node in signalling networks with signal-specific properties.

Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule) form a heterodimeric complex that associates with endosomal membranes and is tyrosine-phosphorylated in response to a variety of growth factors including EGF (epidermal growth factor), HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). Phosphorylation of the Hrs-STAM complex requires receptor endocytosis. We show that an intact UIM (ubiquitin interaction motif) within Hrs is a conserved requirement for Hrs phosphorylation downstream of both EGF and HGF stimulations. Consistent with this, expression of a dominant-negative form of the E3 ubiquitin ligase, c-Cbl, inhibits EGF- and HGF-dependent Hrs phosphorylation. Despite this conservation, kinase inhibitor profiles using PP1 (4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and SU6656 indicate that distinct non-receptor tyrosine kinases couple EGF, HGF and PDGF stimulation with the tyrosine phosphorylation of the Hrs-STAM complex. Crucially, analysis with phospho-specific antibodies indicates that these kinases generate a signal-specific, combinatorial phosphorylation profile of the Hrs-STAM complex, with the potential of diversifying tyrosine kinase receptor signalling through a common element.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Translation of aberrant mRNAs lacking a termination codon or with a shortened 3'-UTR is repressed after initiation in yeast.

A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. Here we report our analysis of translation and decay of nonstop mRNAs in Saccharomyces cerevisiae. Although the reduction of nonstop mRNAs was only 4.5-fold, a level that is sufficient for residual protein synthesis, translation products of nonstop mRNAs were hardly detectable. We show that nonstop mRNAs were associated with polysomes, but not with Pab1p. We also show that ribosomes translating nonstop mRNA formed stable and heavy polysome complexes with mRNA. These data suggest that ribosome stalling at the 3' end of nonstop mRNA may block further rounds of translation, hence repressing protein synthesis. Furthermore, it was found that the 5' --> 3' decay pathway was accelerated for nonstop mRNA decay in the absence of Ski7p. We also found that translation of aberrant mRNAs with a shortened 3'-UTR was repressed, suggesting that an improper spatial distance between the termination codon and the 3' end of mRNA results in translation repression.

3' Untranslated Regions↗
PubMed2005

M-CSF stimulated differentiation requires persistent MEK activity and MAPK phosphorylation independent of Grb2-Sos association and phosphatidylinositol 3-kinase activity.

Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.

Animals↗