The chemokine growth-regulated oncogene-alpha promotes spinal cord oligodendrocyte precursor proliferation

Chemokines, (chemotactic cytokines) are a family of regulatory molecules involved in modulating inflammatory responses. Here we demonstrate that the chemokine growth-regulated oncogene-alpha (GRO-alpha) is a potent promoter of oligodendrocyte precursor proliferation. The proliferative response of immature spinal cord oligodendrocyte precursors to their major mitogen, platelet derived growth factor (PDGF), is dramatically enhanced by GRO-alpha present in spinal cord conditioned medium. One source of GRO-alpha is a subset of spinal cord astrocytes. Cultures of astrocytes contain GRO-alpha mRNA and protein and secrete biologically active concentrations of GRO-alpha. In postnatal spinal cord white matter the location of GRO-alpha-immunoreactive cells is developmentally regulated: GRO-alpha+ cells first appear in ventral and later in dorsal spinal cord white matter. These results suggest that localized proliferation of oligodendrocytes is mediated by synergy between PDGF and GRO-alpha.     

Density-dependent feedback inhibition of oligodendrocyte precursor expansion

The myelin sheath in the vertebrate CNS is formed by oligodendrocytes. The number of oligodendrocytes in a mature axon tract must be sufficient to myelinate all appropriate axons. How the number of oligodendrocytes is matched to axonal requirements and whether such matching involves axon-oligodendrocyte signaling or intrinsic oligodendrocyte self-regulation are not clear. Using a combination of in vitro analyses, we demonstrate that oligodendrocyte precursors closely regulate their numbers through interactions between adjacent precursors. In low-density rat spinal cord cultures, the number of oligodendrocyte lineage cells increases rapidly. The addition of large numbers of oligodendrocyte precursors substantially reduces precursor expansion and results in a normalization of oligodendrocyte lineage cell numbers in the cultures over time. Thus, the number of oligodendrocyte lineage cells that develop appears dependent on the density of oligodendrocyte lineage cells. This normalization of cell number is reflected in assays of clonal potential and proliferation. For example, precursors gave rise to fewer progeny and proliferated less at high density. Reduced precursor expansion at high density was not attributable to the depletion of growth factors. Cocultures of high and low densities did not inhibit precursor expansion in low-density cultures, suggesting the requirement for local cell-cell interactions. The inhibition of precursor expansion was cell-type-specific and dependent on the presence of oligodendrocyte lineage cells. We propose that this density-dependent feedback inhibition of oligodendrocyte precursor expansion may play a primary role in regulating the number of oligodendrocytes in the developing spinal cord.     

Effects of thyroid hormone on embryonic oligodendrocyte precursor cell development in vivo and in vitro

The oligodendrocyte precursor cell divides a limited number of times before terminal differentiation. The timing of differentiation depends on both intracellular mechanisms and extracellular signals, including mitogens that stimulate proliferation and signals such as thyroid hormone (TH) and retinoic acid (RA) that help trigger the cells to stop dividing and differentiate. We show here that, both in vivo and in vitro, TH is required for the normal development of rodent optic nerve oligodendrocytes, although in its absence some oligodendrocyte development still occurs, perhaps promoted by signals from axons. We also demonstrate that TH from both mother and pup plays a part in oligodendrocyte development in vivo. Finally, we show that precursors in embryonic nerve cultures differ from those in postnatal cultures in two ways: they respond much better to TH than to RA, and they respond more slowly to TH, suggesting that oligodendrocyte precursor cells mature during their early development.     

Efficacy and pharmacokinetics of a new intrarectal quinine formulation in children with Plasmodium falciparum malaria

The O-2A progenitor cell, which serves as a stem cell for the myelinating oligodendrocyte, has been implicated as a major target for radiation-induced spinal cord injury. In an attempt to increase the number of O-2A cells in the spinal cord, we applied an ex vivo gene therapy procedure for delivering platelet derived growth factor (PDGF). Recombinant fibroblasts expressing PDGF A chain were injected into the cisterna magna of adult rats, which resulted in cell seeding of the subarachnoid space of the cervical spinal cord. The number of O-2A progenitors in the cervical spinal cord was then assessed with an in vitro clonogenic assay. O-2A cells were found to be increased 8 days after recombinant cell injection, and they remained elevated up to at least 14 days. Analysis of O-2A colonies indicated that the implantation of PDGF-expressing cells increased the number of O-2A progenitors without affecting their in vitro proliferation potential or differentiation capacity. These data suggest that implantation of PDGF-expressing cells in the subarachnoid space of the cervical spinal cord may influence a stem cell population critical to the repair of demyelinated lesions.     

Failure of spinal cord oligodendrocyte development in mice lacking neuregulin

Oligodendrocytes develop from a subpopulation of precursor cells within the ventral ventricular zone of the spinal cord. The molecular cues that direct this spatially and temporally restricted event seem to originate in part from structures ventral to and within the spinal cord. Here, we present evidence that the family of ligands termed neuregulins are necessary for the normal generation of mouse spinal cord oligodendrocytes. Oligodendrocytes mature in spinal cord explants from wild-type mice and mice heterozygotic for a null mutation in the neuregulin gene (NRG +/-) in a temporal sequence of developmental events that replicates that observed in vivo. However, in spinal cord explants derived from mice lacking neuregulin (NRG -/-), oligodendrocytes fail to develop. Addition of recombinant neuregulin to spinal cord explants from NRG -/- mice rescues oligodendrocyte development. In wild-type spinal cord explants, inhibitors of neuregulin mimic the inhibition of oligodendrocyte development that occurs in NRG -/- explants. In embryonic mouse spinal cord, neuregulins are present in motor neurons and the ventral ventricular zone where they likely exert their influence on early oligodendrocyte precursor cells.     

Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells

In the past decade, considerable progress has been made in the understanding of the biology of rodent oligodendrocyte precursor cells and their role in the generation of oligodendrocytes in the developing and adult rodent CNS. Much less is known about human oligodendrocyte lineage cells and about the reasons for the failure of the regeneration of the oligodendrocyte population during chronic stages of multiple sclerosis (MS). In particular, the fate of the oligodendrocyte precursor population in MS has remained elusive. The present study examined the possibility that oligodendrocyte regeneration ultimately fails because of the local destruction of both oligodendrocytes and their precursor cells. Analysis of chronic stage MS tissue suggested that this is not the case, because all chronic MS lesions studied contained significant numbers of oligodendrocyte precursor cells, identified as process-bearing cells that bound the O4 antibody but not antibodies to GalC and GFAP. The oligodendrocyte precursor cells appeared, however, to be relatively quiescent, because none expressed the nuclear proliferation antigen recognized by the Ki-67 antibody, and because most lesions lacked myelinating oligodendrocytes in their centers. Thus, it appears that the regeneration of the oligodendrocyte population fails during chronic stages of MS because of the inability of oligodendrocyte precursor cells to proliferate and differentiate rather than because of the local destruction of all oligodendrocyte lineage cells. The identification of ways of stimulating the endogenous oligodendrocyte precursor population to expand and generate remyelinating cells may represent an alternative to transplantation of oligodendrocyte lineage cells to promote myelin repair in MS.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.     

Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons

To study how an increase in axon number influences the number of glial cells in the mammalian optic nerve, we have analyzed a previously described transgenic mouse that expresses the human bcl-2 gene from a neuron-specific enolase promoter. In these mice, the normal postnatal loss of retinal ganglion cell axons is greatly decreased and, as a consequence, the number of axons in the optic nerve is increased by approximately 80% compared with wild-type mice. Remarkably, the numbers of oligodendrocytes, astrocytes, and microglial cells are all increased proportionally in the transgenic optic nerve. The increase in oligodendrocytes apparently results from both a decrease in normal oligodendrocyte death and an increase in oligodendrocyte precursor cell proliferation, whereas the increase in astrocytes apparently results from an increase in the proliferation of astrocyte lineage cells. Unexpectedly, the transgene is expressed in oligodendrocytes and astrocytes, but this does not seem to be responsible for the increased numbers of these cells. These findings indicate that developing neurons and glial cells can interact to adjust glial cell numbers appropriately when neuronal numbers are increased. We also show that the expression of the bcl-2 transgene in retinal ganglion cells protects the cell body from programmed cell death when the axon is cut, but it does not protect the isolated axon from Wallerian degeneration, even though the transgene-encoded protein is present in the axon.     

NT-3, but not BDNF, prevents atrophy and death of axotomized spinal cord projection neurons

Following spinal cord injury, projection neurons are frequently axotomized and many of the cells subsequently die. One goal in spinal injury research is to preserve damaged neurons so that ultimately they are accessible to regeneration-promoting strategies. Here we ask if neurotrophin treatment can prevent atrophy and death of axotomized sensory projection neurons. In adult rats, a hemisection was made in the thoracic spinal cord and axotomized neurons were retrogradely labelled with Fluoro-Gold. Four distinct populations of cells were identified in the lumbar spinal cord, and both numbers and sizes of labelled cells were assessed at different time points postlesion. A progressive and significant degeneration was observed over time with severe atrophy apparent in all cell populations and significant cell loss evident by 4 weeks postlesion. This time point was used to assess neurotrophin effects. Hemisected rats were treated with either neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF, 12 microg/day for each), or a vehicle solution, delivered continuously to the lesion site via an osmotic minipump. Treatment with NT-3, but not BDNF, completely reversed cell atrophy in three of the four cell populations and also induced a significant increase in the number of surviving cells. In situ hybridization experiments showed trkB and trkC mRNA to be expressed in the majority of ascending spinal projection neurons, suggesting that these cells should be responsive to both BDNF and NT-3. However, only NT-3 treatment was neuroprotective, indicating that BDNF may not have reached the cell bodies of injured neurons. These results demonstrate that NT-3 may be of benefit in preventing the secondary cell loss that occurs following spinal injury.     

Purification and characterization of adult oligodendrocyte precursor cells from the rat optic nerve

Oligodendrocyte precursor cells (OPCs) persist in substantial numbers in the adult brain in a quiescent state suggesting that they may provide a source of new oligodendrocytes after injury. To determine whether adult OPCs have the capacity to divide rapidly, we have developed a method to highly purify OPCs from adult optic nerve and have directly compared their properties with their perinatal counterparts. When cultured in platelet-derived growth factor (PDGF), an astrocyte-derived mitogen, perinatal OPCs divided approximately once per day, whereas adult OPCs divided only once every 3 or 4 d. The proliferation rate of adult OPCs was not increased by addition of fibroblast growth factor (FGF) or of the neuregulin glial growth factor 2 (GGF2), two mitogens that are normally produced by retinal ganglion cells. cAMP elevation has been shown previously to be essential for Schwann cells to survive and divide in response to GGF2 and other mitogens. Similarly we found that when cAMP levels were elevated, GGF2 alone was sufficient to induce perinatal OPCs to divide slowly, approximately once every 4 d, but adult OPCs still did not divide. When PDGF was combined with GGF2 and cAMP elevation, however, the adult OPCs began to divide rapidly. These findings indicate that adult OPCs are intrinsically different than perinatal OPCs. They are not senescent cells, however, because they retain the capacity to divide rapidly. Thus, after demyelinating injuries, enhanced axonal release of GGF2 or a related neuregulin might collaborate with astrocyte-derived PDGF to induce rapid division of adult OPCs.     

Age-dependent differences in the effects of GDNF and NT-3 on the development of neurons and glia from neural crest-derived precursors immunoselected from the fetal rat gut: expression of GFRalpha-1 in vitro and in vivo

No enteric neurons or glia develop in the gut below the rostral foregut in mice lacking glial cell line-derived neurotrophic factor (GDNF) or Ret. We analyzed the nature and age dependence of the effects of GDNF and, for comparison, those of NT-3, on the in vitro development of the precursors of enteric neurons and glia. Positive and negative immunoselection with antibodies to p75(NTR) were used to isolate crest-derived and crest-depleted populations of cells from the fetal rat bowel at E12, 14, and 16. Cells were typed immunocytochemically. GDNF stimulated the proliferation of nestin-expressing precursor cells isolated at E12, but not at E14-16. GDNF promoted the development of peripherin-expressing neurons (E12 > E14-16) and expression of TrkC. GDNF inhibited expression of S-100-expressing glia at E14-16. NT-3 did not affect cells isolated at E12, never stimulated precursors to proliferate, and promoted glial as well as neuronal development at E14-16. GFRalpha-1 was expressed both by crest- and non-crest-derived cells, although only crest-derived cells anchored GFRalpha-1 and GFRalpha-2 (GFRalpha-1 > GFRalpha-2). GDNF increased the number of neurons anchoring GFRalpha-1. GFRalpha-1 is immunocytochemically detectable in neurons of the E13 intestine and persists in adult neurons of both plexuses. We suggest that GDNF stimulates the proliferation of an early (E12) NT-3-insensitive precursor common to enteric neurons and glia; by E14, this common precursor is replaced by specified NT-3-responsive neuronal and glial progenitors. GDNF exerts a neurotrophic, but not a mitogenic, effect on the neuronal progenitor. The glial progenitor is not maintained by GDNF.     

Neurotrophin-3 antibodies disrupt the normal development of the chick retina

When chick embryos are treated with a monoclonal antibody specifically blocking the activity of neurotrophin-3 (NT-3), the development of the retina is profoundly affected. Fewer axons are found in the optic nerve, and the retina shows abnormalities in all layers. Early during retinogenesis, the proportion of dividing cells is higher in NT-3-deprived embryos compared with age-matched controls and that of differentiated neurons is smaller. The NT-3 receptor trkC is expressed early by a majority of retinal cells, and NT-3 is present in the retina at the earliest stage studied. Initially, it is located mainly in the pigmented epithelium, with a shift toward the neural retina as development proceeds. Thus, NT-3 seems to be an essential intrinsic signal acting early in development to promote the differentiation and survival of many retinal neurons.     

Crk protein binds to PDGF receptor and insulin receptor substrate-1 with different modulating effects on PDGF- and insulin-dependent signaling pathways

We have studied the involvement of murine c-Crk, an SH2/SH3 containing adaptor protein, in signaling pathways stimulated by different receptor tyrosine kinases. We show here that c-Crk is associated with components of insulin- and PDGF-dependent signaling pathways. Insulin treatment of murine myoblast cells induces the formation of stable complex of endogenous c-Crk with insulin receptor substrate-1 (IRS-1) mediated via the SH2 domain of Crk. The ligand dependent physical association of c-Crk with IRS-1 is direct. However IRS-1 is also co-precipitated with c-Crk from quiescent L6 cells. The association of IRS-1 with c-Crk in quiescent cells is probably not direct since Far Western blot analysis did not reveal the binding of neither SH2 domain nor amino-terminal SH3 domain of c-Crk to IRS-1 from unstimulated cells. We also show that PDGF treatment of murine myoblast cells induces association of c-Crk with the PDGF receptor and tyrosine phosphorylation of c-Crk. Overexpression of c-Crk enhanced insulin- but not PDGF-induced activation of MAP kinases when compared to parental cell lines. Thus, the formation of the direct IRS-1/Crk complex appears to be crucial for Crk-mediated insulin-induced activation of MAP kinase, whereas Crk is probably involved in other PDGF-induced responses. These data provide support to the hypothesis that insulin and PDGF employ different mechanisms for activation of MAP kinase cascade.     

Mitochondrial malic enzyme: purification from bovine brain, generation of an antiserum, and immunocytochemical localization in neurons of rat brain

The receptor kinase activity associated with the epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor plays an important role in ligand-induced signaling events. The effect of specific, synthetic chemical inhibitors of PDGF- and EGF-mediated receptor tyrosine autophosphorylation on receptor signaling were examined in NIH 3T3 cells overexpressing PDGF or EGF receptors. Specific inhibition of ligand-dependent receptor autophosphorylation, PI3K activation, mitogen-activated protein kinase (MAPK) activation, cyclin E-associated kinase activity and cell proliferation was measured after treatment of cells with these inhibitors. A synthetic PDGF receptor kinase inhibitor exhibited specific inhibitory properties when tested for PDGF-induced receptor autophosphorylation, MAPK activity, PI3K activation, entry into S phase and cyclin E-associated kinase activity. A synthetic EGF receptor kinase inhibitor showed selective inhibitor properties when tested for EGF-induced receptor autophosphorylation, MAPK activation, PI3K activation, entry into S phase and cyclin E-associated kinase activity. In both cases, these compounds were found to be effective as inducers of growth arrest and accumulation of cells in the G1 phase of the cell cycle after ligand treatment. However, at high concentrations, the EGF receptor kinase inhibitor was observed to exhibit some nonspecific effects as demonstrated by attenuation of PDGF-induced receptor autophosphorylation and cell cycle progression. This demonstrates that it is critical to use the lowest concentration of such an inhibitor that will alter the response under investigation, to have confidence that the conclusions derived from the use of such inhibitor are valid. We conclude that these experimental parameters signify useful end points to measure the relative selectivity of tyrosine kinase inhibitors that affect receptor-mediated signal transduction.     

Neurotrophins in the developing and regenerating visual system

The neurotrophins NGF, BDNF, NT-3 and NT-4 have a wide range of effects in the development and regeneration of neural circuits in the visual system of vertebrates. This review focuses on the localization and functions of neurotrophins in the retina, lateral geniculate nucleus, suprachiasmatic nucleus, superior colliculus/optic tectum, and isthmic nuclei. Research of the past 20 years has shown that neurotrophins and their receptors are localized in numerous visual centers from the retina to the visual cortex, and that neurotrophins influence proliferation, neurite outgrowth and survival of cells in the visual system in vitro and in vivo. A relationship between electrical activity and neurotrophic functions has been established in several visual centers in the CNS, and neurotrophins have been implicated in synaptic plasticity in the visual cortex. Besides functions of neurotrophins as retrograde, target-derived trophic factors, recent data indicate that neurotrophins may have anterograde, afferent as well as local, paracrine actions in the retina, optic nerve and the visual cortex. Some neurotrophins appear to regulate proliferation and survival of glial cells in the optic pathways. Neurotrophins increase the survival of retinal ganglion cells after axotomy or ischemia and they promote the regeneration of retinal ganglion cell axons in some vertebration. Neurotrophins also rescue photoreceptors from degeneration. These findings implicate the neurotrophins not only as important regulators during development, but also as potential therapeutic agents in degenerative retinal diseases and after optic nerve injury.     

Embryonic mesoderm cells spread in response to platelet-derived growth factor and signaling by phosphatidylinositol 3-kinase

Abnormal mesoderm movement, leading to defects in axial organization, is observed in mouse and Xenopus laevis embryos deprived of platelet-derived growth factor (PDGF) AA signaling. However, neither the cellular response to PDGF nor the signaling pathways involved are understood. Herein we describe an in vitro assay to examine the direct effect of PDGF AA on aggregates of Xenopus embryonic mesoderm cells. We find that PDGF AA stimulates aggregates to spread on fibronectin. This behavior is similar to that of migrating mesoderm cells in vivo that spread and form lamellipodia and filipodia on contact with fibronectin-rich extracellular matrix. We go on to show two lines of evidence that implicate phosphatidylinositol 3-kinase (PI3K) as an important component of PDGF-induced mesoderm cell spreading. (i) The fungal metabolite wortmannin, which inhibits signaling by PI3K, blocks mesoderm spreading in response to PDGF AA. (ii) Activation of a series of receptors with specific tyrosine-to-phenylalanine mutations revealed PDGF-induced spreading of mesoderm cells depends on PI3K but not on other signaling molecules that interact with PDGF receptors including phospholipase C gamma, Ras GTPase-activating protein, and phosphotyrosine phosphatase SHPTP2. These results indicate that a PDGF signal, medicated by PI3K, can facilitate embryonic mesoderm cell spreading on fibronectin. We propose that PDGF, produced by the ectoderm, influences the adhesive properties of the adjacent mesoderm cells during gastrulation.     

Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury

The injured adult mammalian spinal cord shows little spontaneous recovery after injury. In the present study, the contribution of projections in the dorsal half of the spinal cord to functional loss after adult spinal cord injury was examined, together with the effects of transgenic cellular delivery of neurotrophin-3 (NT-3) on morphological and functional disturbances. Adult rats underwent bilateral dorsal column spinal cord lesions that remove the dorsal corticospinal projections or underwent more extensive resections of the entire dorsal spinal cord bilaterally that remove corticospinal, rubrospinal, and cerulospinal projections. Long-lasting functional deficits were observed on a motor grid task requiring detailed integration of sensorimotor skills, but only in animals with dorsal hemisection lesions as opposed to dorsal column lesions. Syngenic primary rat fibroblasts genetically modified to produce NT-3 were then grafted to acute spinal cord dorsal hemisection lesion cavities. Up to 3 months later, significant partial functional recovery occurred in NT-3-grafted animals together with a significant increase in corticospinal axon growth at and distal to the injury site. These findings indicate that (1) several spinal pathways contribute to loss of motor function after spinal cord injury, (2) NT-3 is a neurotrophic factor for the injured corticospinal projection, and (3) functional deficits are partially ameliorated by local cellular delivery of NT-3. Lesions of the corticospinal projection may be necessary, but insufficient in isolation, to cause sensorimotor dysfunction after spinal cord injury in the rat.     

The proliferation of mature oligodendrocytes in vitro is stimulated by basic fibroblast growth factor and inhibited by oligodendrocyte-type 2 astrocyte precursors

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are mitogens for bipotential oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. We investigated the mitogenic effect of these growth factors on quiescent mature oligodendrocytes (OL) expressing myelin basic protein (MBP) in OL cultures that were treated for 3 days with cytosine arabinoside (ARA-C) in order to kill O-2A precursors which divide in chemically defined medium. After treatment with ARA-C proliferation decreased and O-2A precursors identified with A2B5 monoclonal antibody were nearly undetectable. After exposure of mature OL to bFGF, cell proliferation increased markedly within 24 hr. PDGF had a much weaker effect. Cultures treated with ARA-C for 3 days and then with bFGF for the next 24 hr and incubated with BrdU for the last 2 hr before the end of the experiment were immunolabeled with anti-MBP or A2B5 and anti-bromodeoxyuridine (BrdU) antibodies. Eighty-seven percent of the cells were MBP+, 10% were both MBP+ and BrdU+, and none was A2B5+ BrdU+, showing that at least a part of the population of mature MBP+ OL retains the ability to reenter the cell cycle in vitro. Since mature OL did not proliferate in response to bFGF in the cultures not treated with ARA-C, i.e., in the presence of O-2A progenitors, we assumed that these precursors were responsible for the lack of mitogenic effect of bFGF on MBP+ OL in such conditions. Conditioned medium from O-2A precursors almost halved the bFGF-induced OL proliferation after treatment with ARA-C, suggesting that O-2A progenitors control the proliferation of a subpopulation of mature OL (possibly young mature OL) via the secretion of active molecule(s).     

APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis

Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.