Independent mechanisms account for the regulation by protein kinase C of the epidermal growth factor receptor affinity and tyrosine-protein kinase activity

The epidermal growth factor (EGF) receptor is a substrate for phosphorylation by the calcium- and phospholipid-dependent protein kinase (protein kinase C) at Thr654. The hypothesis that this phosphorylation is causally related to the regulation of the functional properties of the EGF receptor was tested by substitution of Thr654 with an alanine residue. Activation of protein kinase C using phorbol ester caused a decrease in the high affinity binding of EGF to Chinese hamster ovary cells expressing wild-type [Thr654]EGF receptors. Similar results were obtained with cells expressing mutated [Ala654]EGF receptors. The regulation of the protein kinase activity of the EGF receptor by protein kinase C was examined using a synthetic peptide substrate for tyrosine phosphorylation. Protein kinase C caused a Ca2+-dependent decrease in the tyrosine-protein kinase activity of the wild-type [Thr654]EGF receptor. In contrast, no inhibition of the tyrosine-protein kinase activity of the mutated [Ala654]EGF receptor caused by protein kinase C was detected. In further experiments, the desensitization of EGF action caused by the activation of protein kinase C was examined by investigating the regulation of the transferrin receptor by EGF. Phorbol ester was observed to cause the desensitization of signaling by the wild-type [Thr654] and mutated [Ala654]EGF receptors. These data are consistent with a role for the phosphorylation of EGF receptor Thr654 in the regulation of the receptor tyrosine-protein kinase activity. However, the inhibition of the high affinity binding of EGF to cell-surface receptors caused by protein kinase C does not require Thr654. It is concluded that independent mechanisms account for the regulation by protein kinase C of the EGF receptor affinity and tyrosine-protein kinase activity.     

Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages.     

Protein kinase C inhibition of the epidermal growth factor receptor tyrosine protein kinase activity is independent of the oligomeric state of the receptor

Treatment of A431 human epidermoid carcinoma cells with 4-phorbol 12-myristate 13-acetate (PMA) causes an inhibition of the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an inhibition of the EGF receptor tyrosine protein kinase activity. The hypothesis that PMA controls EGF receptor function by regulating the oligomeric state of the receptor was tested. Dimeric EGF receptors bound to 125I-EGF were identified by covalent cross-linking analysis using disuccinimidyl suberimidate. Treatment of cells with PMA in the presence of 20 nM 125I-EGF caused no significant change in the level of labeled cross-linked monomeric and dimeric receptor species. Investigation of the in vitro autophosphorylation of receptor monomers and dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide demonstrated that the treatment of cells with PMA caused an inhibition of the tyrosine phosphorylation of both monomeric and dimeric EGF receptors. We conclude that the inhibition of the EGF receptor tyrosine protein kinase activity caused by PMA is not associated with the regulation of the oligomeric state of the EGF receptor.     

Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

Desensitization of the isolated beta 2-adrenergic receptor by beta-adrenergic receptor kinase, cAMP-dependent protein kinase, and protein kinase C occurs via distinct molecular mechanisms.

Exposure of beta 2-adrenergic receptors (beta 2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta 2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta ARK phosphorylation of beta 2AR when assayed in the presence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.     

Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ～300 ? distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios. The IC(50) of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC(50) if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289-12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.     

Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line.

Metabolic labeling of simian virus 40-immortalized murine macrophages with 32Pi and immunoblotting with antibodies to phosphotyrosine demonstrated that the c-fms proto-oncogene product (colony-stimulating factor 1 [CSF-1] receptor) was phosphorylated on tyrosine in vivo and rapidly degraded in response to CSF-1. Stimulation of the CSF-1 receptor also induced immediate phosphorylation of several other cellular proteins on tyrosine. By contrast, the mature cell surface glycoprotein encoded by the v-fms oncogene was phosphorylated on tyrosine in the absence of CSF-1, suggesting that it functions as a ligand-independent kinase.     

Phenobarbital decreases hepatocyte EGF receptor expression independent of protein kinase C activation

The liver tumor promoter, phenobarbital, directly applied to cultured, adult rat hepatocytes at concentrations of greater than 1 mM, decreases cellular surface binding of EGF. This effect of phenobarbital resembles that of 4 beta-phorbol-12 alpha-myristate-13 beta-acetate (TPA) in that both decrease EGF receptor number, but do not affect receptor affinity. The effects of the two tumor promoters differ however, in that only TPA reduces high affinity EGF binding by A431 cells. They also differ in that TPA, but not phenobarbital, causes redistribution of protein kinase C from a soluble to a membranous hepatocyte subcellular fraction. These data indicate that decreased EGF binding is a common hepatocyte response to the tumor promoters, TPA and phenobarbital, but that this response can be mediated by either a TPA-activated, protein kinase C-dependent pathway or by a phenobarbital-sensitive, protein kinase C-independent pathway.     

Colony-stimulating factor 1 activates protein kinase C in human monocytes.

Colony-stimulating factor 1 (CSF-1) is required for the survival, proliferation and differentiation of monocytes. We previously demonstrated that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein and that the induction of Na+ influx by CSF-1 is a pertussis toxin-sensitive event. The present studies have examined activation of protein kinase C as a potential intracellular signaling event induced by the activated CSF-1 receptor. The results demonstrate that CSF-1 stimulates translocation of protein kinase C activity from the cytosol to membrane fractions. This activation of protein kinase C was sensitive to pretreatment of the monocytes with pertussis toxin. Lipid distribution studies demonstrated that phosphatidylcholine (PC) is the major phospholipid in human monocytes. Moreover, the results indicate that CSF-1 stimulation is associated with decreases in PC, but not in phosphatidylinositol (PI), levels. The absence of an effect of CSF-1 on PI turnover was confirmed by the lack of changes in inositol phosphate production. In contrast, CSF-1 stimulation was associated with increased hydrolysis of PC to phosphorylcholine and diacylglycerol (DAG) in both intact monocytes and cell-free assays. Furthermore, the increase in PC turnover induced by CSF-1 was sensitive to pertussis toxin. The results also demonstrate that the induction of Na+ influx by CSF-1 is inhibited by the protein kinase C inhibitors staurosporine and the isoquinoline derivative H7, but not by HA1004.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C phosphorylation at Thr 654 of the unoccupied EGF receptor and EGF binding regulate functional receptor loss by independent mechanisms

To test the functional consequence of phosphorylation of the EGF receptor at Thr 654 by protein kinase C, the normal Thr 654 human EGF receptor cDNA or a mutant encoding an Ala 654 were expressed in heterologous cells. In cell lines expressing both the Thr 654 and Ala 654 receptors, functional cell-surface Thr 654 receptors were reduced or were totally lost, but were not degraded, following activation of protein kinase C by phorbol esters (TPA), whereas Ala 654 receptors were unaffected. These data suggest that protein kinase C regulates ligand-independent receptor binding and internalization via phosphorylation of Thr 654 of the EGF holoreceptor. Because EGF induces internalization and degradation of the Ala 654 EGF receptor, at least two independent mechanisms can serve to signal loss of functional EGF receptors.     

Protein kinase C-dependent and -independent mechanisms regulating the parotid substance P receptor as revealed by differential effects of protein kinase C inhibitors.

Substance P-induced inositol trisphosphate (InsP3) formation was inhibited by 1 microM-4 beta-phorbol 12,13-dibutyrate (PDBu) in rat parotid acinar cells. The inhibitory effect of PDBu was reversed by the protein kinase C inhibitors H-7 or K252a. Substance P also elicits a persistent desensitization of subsequent substance P-stimulated InsP3 formation. However, this desensitization was not inhibited by H-7. In addition, H-7 had no effect on the time course of substance P-induced InsP3 formation. These results suggest that, although activation of protein kinase C by phorbol esters can inhibit the substance P receptor-linked phospholipase C pathway, this mechanism apparently plays little, if any, role in regulating this system after activation by substance P.     

Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated by PC-derived diacylglycerol, as a Raf activator. A dominant negative mutant of PKC epsilon inhibited both proliferation of NIH 3T3 cells and activation of Raf in COS cells. Conversely, overexpression of active PKC epsilon stimulated Raf kinase activity in COS cells and overcame the inhibitory effects of dominant negative Ras in NIH 3T3 cells. PKC epsilon also stimulated Raf kinase in baculovirus-infected Spodoptera frugiperda Sf9 cells and was able to directly activate Raf in vitro. Consistent with its previously reported activity as a Raf activator in vitro, PKC alpha functioned similarly to PKC epsilon in both NIH 3T3 and COS cell assays. In addition, constitutively active mutants of both PKC alpha and PKC epsilon overcame the inhibitory effects of dominant negative mutants of the other PKC isotype, indicating that these diacylglycerol-regulated PKCs function as redundant activators of Raf-1 in vivo.     

Mitogen-activated protein kinase phosphatase PHS1 is retained in the cytoplasm by nuclear extrusion signal-dependent and independent mechanisms

The organization of plant microtubule arrays is thought to be regulated by phosphorylation and other signaling cascades, but the molecular components involved are largely unknown. We have previously found that a dominant missense mutation (phs1-1) in a putative kinase-docking motif of an Arabidopsis PHS1 phosphatase, which belongs to the mitogen-activated protein kinase phosphatase (MKP) family, compromises the stability of cortical microtubules. We here report that suppressor screening of phs1-1 recovered several intragenic recessive mutations in PHS1. In contrast to our previous report, null alleles of PHS1 were almost indistinguishable from the wild type in morphology, but their roots skewed to the abnormal direction when grew in the presence of low doses of a microtubule-destabilizing drug. PHS1 is mainly expressed in elongating cells, where the protein was distributed in the cytoplasm, predominantly in a microsomal fraction. Recruitment of green fluorescent protein-tagged PHS1 in endomembrane aggregates after treatment with brefeldin A or in an endomembrane-organization mutant suggests that an association with endomembranes retains PHS1 in the cytoplasm. A nuclear export signal identified in the C-terminal tail also contributes to the robust cytoplasmic retention of PHS1.     

Parathyroid hormone receptor internalization is independent of protein kinase A and phospholipase C activation

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) binding to their common receptor stimulates second messenger accumulation, receptor phosphorylation, and internalization. LLC-PK(1) cells expressing a green fluorescent protein-tagged PTH/PTHrP receptor show time- and dose-dependent receptor internalization. The internalized receptors colocalize with clathrin-coated pits. Internalization is stimulated by PTH analogs that bind to and activate the PTH/PTHrP receptor. Cell lines expressing a mutant protein kinase A regulatory subunit that is resistant to cAMP and/or a mutant receptor (DSEL mutant) that does not activate phospholipase C internalize their receptors normally. In addition, internalization of the wild-type receptor and the DSEL mutant is stimulated by the PTH analog [Gly(1),Arg(19)]hPTH-(1-28), which does not stimulate phospholipase C. Forskolin, IBMX, and the active phorbol ester, phorbol-12-myristate-13-acetate, did not promote receptor internalization or increase PTH-induced internalization. These data indicate that ligand-induced internalization of the PTH/PTHrP receptor requires both ligand binding and receptor activation but does not involve stimulation of adenylate cyclase/protein kinase A or phospholipase C/protein kinase C.     

Dual regulation of EDG1/S1P(1) receptor phosphorylation and internalization by protein kinase C and G-protein-coupled receptor kinase 2.

Here we demonstrate that phosphorylation of the sphingosine 1-phosphate (SSP) receptor "endothelial differentiation gene 1" (EDG1 or S1P(1)) receptor is increased in response to either SSP or phorbol 12-myristate 13-acetate (PMA) exposure but not lysophosphatidic acid. Phosphoamino acid analysis demonstrated that SSP stimulated the accumulation of phosphoserine and phosphothreonine but not phosphotyrosine. An inhibitor of PMA-stimulated EDG1 phosphorylation failed to block SSP-stimulated phosphorylation. Additionally, removal of 12 amino acids from the carboxyl terminus of EDG1 specifically reduced SSP- but not PMA-stimulated phosphorylation, suggesting that SSP and PMA increase EDG1 phosphorylation via distinct mechanisms. In vitro assays revealed that G-protein-coupled receptor kinase 2 may be at least partially responsible for SSP-stimulated EDG1 phosphorylation observed in intact cells. In addition, phosphorylation by PMA and SSP were associated with a loss of EDG1 from the cell surface by distinct mechanisms. Removal of 12 residues from the carboxyl terminus of EDG1 completely inhibited SSP-mediated internalization, suggesting that this domain dictates susceptibility to receptor internalization while retaining sensitivity to SSP-stimulated phosphorylation. Thus, we conclude that (a) EDG1 phosphorylation and internalization are controlled via independent mechanisms by agonist occupation of the receptor and protein kinase C activation, and (b) although determinants within the receptor's carboxyl-terminal tail conferring EDG1 sensitivity to agonist-mediated internalization and G-protein-coupled receptor kinase phosphorylation exhibit a degree of overlap, the two phenomena are separable.     

Constitutive phosphorylation by protein kinase C regulates D1 dopamine receptor signaling

The D(1) dopamine receptor (D(1) DAR) is robustly phosphorylated by multiple protein kinases, yet the phosphorylation sites and functional consequences of these modifications are not fully understood. Here, we report that the D(1) DAR is phosphorylated by protein kinase C (PKC) in the absence of agonist stimulation. Phosphorylation of the D(1) DAR by PKC is constitutive in nature, can be induced by phorbol ester treatment or through activation of Gq-mediated signal transduction pathways, and is abolished by PKC inhibitors. We demonstrate that most, but not all, isoforms of PKC are capable of phosphorylating the receptor. To directly assess the functional role of PKC phosphorylation of the D(1) DAR, a site-directed mutagenesis approach was used to identify the PKC sites within the receptor. Five serine residues were found to mediate the PKC phosphorylation. Replacement of these residues had no effect on D(1) DAR expression or agonist-induced desensitization; however, G protein coupling and cAMP accumulation were significantly enhanced in PKC-null D(1) DAR. Thus, constitutive or heterologous PKC phosphorylation of the D(1) DAR dampens dopamine activation of the receptor, most likely occurring in a context-specific manner, mediated by the repertoire of PKC isozymes within the cell.     

New signal transduction mechanisms of atrial natriuretic factor: inhibition of phosphorylation of protein kinase C and A 240 kDa protein in adrenal cortical plasma membrane by cGMP dependent and independent mechanisms

The effects of synthetic atrial natriuretic factor (ANF) on the state of protein phosphorylation in plasma membranes of bovine adrenal cortex have been studied in vitro. ANF (1x10(-8)M - 1x10(-7)M) specifically inhibited the phosphorylation of two distinct proteins of 78 kDa and 240 kDa. Immunoblotting with specific antiserum to protein kinase C produced evidence that 78 kDa protein is most likely the protein kinase C whose phosphorylation is inhibited by both ANF and cGMP. However, cGMP did not affect the phosphorylation of 240 kDa protein, indicating a new cGMP-independent mechanism of ANF action in the adrenal, which is compatible with the lack of action of cGMP and its analogs in ANF-induced inhibition of aldosterone secretion from adrenal cortex. The inhibition of phosphorylation of putative protein kinase C by ANF or cGMP indicates a hitherto unknown signal transduction mechanism of ANF.     

Ligand dependent and independent internalization and nuclear translocation of fibroblast growth factor (FGF) receptor 1

Basic fibroblast growth factor (FGF-2) is one of the prototype members of a rapidly expanding family of polypeptides. FGF-2 acts on cells via a dual-receptor system consisting of high-affinity tyrosine kinase receptors (FGFR) and low-affinity receptors comprised of heparan sulfate proteoglycans. Following ligand binding and subsequent internalization, both FGF-2 and FGFR1 are translocated to the nucleus where they have activities distinct from those expressed at the cell surface. Despite the growing number of growth factors and receptors shown to translocate to the nucleus, little is known about the mechanisms of internalization and translocation and how these processes are regulated. In the studies reported in this paper, we examined the roles of clathrin-dependent and -independent endocytosis in the uptake of FGFR1 and one of its ligands, FGF-2. While the uptake of FGF-2 occurred at least partly by a caveolar-dependent mechanism, that of FGFR1 was independent of both caveolae and coated pits. Surprisingly, neither the uptake of FGF-2 nor FGFR1 required the activity of the receptor tyrosine kinase. In addition, we identified a cell cycle-dependent pathway of FGFR1 nuclear translocation that appears to be independent of ligand binding.     

Apoprotein A-1 is a cofactor independent substrate of protein kinase C

Apoprotein A-1 (apo A-1), the predominant protein constituent of high density lipoproteins (HDL), was phosphorylated by protein kinase C (PKC). Optimal phosphorylation of lipid-free apo A-1 occurs in the absence of calcium, phosphatidyl serine (PS), and diolein (DO). However, HDL-bound apo A-1 was not phosphorylated by PKC. Furthermore, addition of either native or reconstituted HDL particles to lipid-free apo A-1 resulted in a concentration-dependent inhibition of phosphorylation. It appears that the phosphorylatable sites on apo A-1 are involved in hydrophobic interaction with the lipids of HDL. Apo A-1 is a novel substrate of PKC because it does not require calcium and lipid cofactors for optimal phosphorylation.     

Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.