Receptor-type protein-tyrosine phosphatase ζ is a functional receptor for interleukin-34.

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.

Macrophage proliferation is regulated through CSF-1 receptor tyrosines 544, 559, and 807.

Colony-stimulating factor-1 (CSF-1)-stimulated CSF-1 receptor (CSF-1R) tyrosine phosphorylation initiates survival, proliferation, and differentiation signaling pathways in macrophages. Either activation loop Y807F or juxtamembrane domain (JMD) Y559F mutations severely compromise CSF-1-regulated proliferation and differentiation. YEF, a CSF-1R in which all eight tyrosines phosphorylated in the activated receptor were mutated to phenylalanine, lacks in vitro kinase activity and in vivo CSF-1-regulated tyrosine phosphorylation. The addition of Tyr-807 alone to the YEF backbone (Y807AB) led to CSF-1-independent but receptor kinase-dependent proliferation, without detectable activation loop Tyr-807 phosphorylation. The addition of Tyr-559 alone (Y559AB) supported a low level of CSF-1-independent proliferation that was slightly enhanced by CSF-1, indicating that Tyr-559 has a positive Tyr-807-independent effect. Consistent with the postulated autoinhibitory role of the JMD Tyr-559 and its relief by ligand-induced Tyr-559 phosphorylation, the addition of Tyr-559 to the Y807AB background suppressed proliferation in the absence of CSF-1, but restored most of the CSF-1-stimulated proliferation. Full restoration of kinase activation and proliferation required the additional add back of JMD Tyr-544. Inhibitor experiments indicate that the constitutive proliferation of Y807AB macrophages is mediated by the phosphatidylinositol 3-kinase (PI3K) and ERK1/2 pathways, whereas proliferation of WT and Y559,807AB macrophages is, in addition, contributed to by Src family kinase (SFK)-dependent pathways. Thus Tyr-807 confers sufficient kinase activity for strong CSF-1-independent proliferation, whereas Tyr-559 maintains the receptor in an inactive state. Tyr-559 phosphorylation releases this restraint and may also contribute to the CSF-1-regulated proliferative response by activating Src family kinase.

Measurement of macrophage growth and differentiation.

This unit provides protocols for measuring the abundance and growth of macrophage precursors in agar cultures and the proliferation of isolated mature macrophages in vitro, by either direct cell counting or by DNA measurement. Methods for the immunohistochemical identification of macrophages and the determination of their proliferative status in vivo by immunofluorescence are also included. It also describes methods for characterization of macrophage differentiation through the immunofluorescence analysis of cell-surface expression of CSF-1 receptor.

A CSF-1 receptor phosphotyrosine 559 signaling pathway regulates receptor ubiquitination and tyrosine phosphorylation.

Receptor tyrosine kinase (RTK) activation involves ligand-induced receptor dimerization and transphosphorylation on tyrosine residues. Colony-stimulating factor-1 (CSF-1)-induced CSF-1 receptor (CSF-1R) tyrosine phosphorylation and ubiquitination were studied in mouse macrophages. Phosphorylation of CSF-1R Tyr-559, required for the binding of Src family kinases (SFKs), was both necessary and sufficient for these responses and for c-Cbl tyrosine phosphorylation and all three responses were inhibited by SFK inhibitors. In c-Cbl-deficient macrophages, CSF-1R ubiquitination and tyrosine phosphorylation were substantially inhibited. Reconstitution with wild-type, but not ubiquitin ligase-defective C381A c-Cbl rescued these responses, while expression of C381A c-Cbl in wild-type macrophages suppressed them. Analysis of site-directed mutations in the CSF-1R further suggests that activated c-Cbl-mediated CSF-1R ubiquitination is required for a conformational change in the major kinase domain that allows amplification of receptor tyrosine phosphorylation and full receptor activation. Thus the results indicate that CSF-1-mediated receptor dimerization leads to a Tyr-559/SFK/c-Cbl pathway resulting in receptor ubiquitination that permits full receptor tyrosine phosphorylation of this class III RTK in macrophages.

Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells.

CSF-1 is broadly expressed and regulates macrophage and osteoclast development. The action and expression of IL-34, a novel CSF-1R ligand, were investigated in the mouse. As expected, huIL-34 stimulated macrophage proliferation via the huCSF-1R, equivalently to huCSF-1, but was much less active at stimulating mouse macrophage proliferation than huCSF-1. Like muCSF-1, muIL-34 and a muIL-34 isoform lacking Q81 stimulated mouse macrophage proliferation, CSF-1R tyrosine phosphorylation, and signaling and synergized with other cytokines to generate macrophages and osteoclasts from cultured progenitors. However, they respectively possessed twofold and fivefold lower affinities for the CSF-1R and correspondingly, lower activities than muCSF-1. Furthermore, muIL-34, when transgenically expressed in a CSF-1-dependent manner in vivo, rescued the bone, osteoclast, tissue macrophage, and fertility defects of Csf1(op)/(op) mice, suggesting similar regulation of CSF-1R-expressing cells by IL-34 and CSF-1. Whole-mount IL34 in situ hybridization and CSF-1 reporter expression revealed that IL34 mRNA was strongly expressed in the embryonic brain at E11.5, prior to the expression of Csf1 mRNA. QRT-PCR revealed that compared with Csf1 mRNA, IL34 mRNA levels were lower in pregnant uterus and in cultured osteoblasts, higher in most regions of the brain and heart, and not compensatorily increased in Csf1(op/op) mouse tissues. Thus, the different spatiotemporal expression of IL-34 and CSF-1 allows for complementary activation of the CSF-1R in developing and adult tissues.

Rapid detergent removal from peptide samples with ethyl acetate for mass spectrometry analysis.

Detergents are required for the extraction of hydrophobic proteins and for the maintenance of their solubility in solution. However, the presence of detergents in the peptide samples severely suppresses ionization in mass spectrometry (MS) analysis and decreases chromatographic resolution in LC-MS. Thus, detergents must be removed for sensitive detection of peptides by MS. This unit describes a rapid protocol in which ethyl acetate extraction is used to remove octylglucoside from protease digests without loss of peptides. This procedure can also be used to reduce interference by sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 in peptide samples for MS analysis.

Primed innate immunity leads to autoinflammatory disease in PSTPIP2-deficient cmo mice.

The mouse Lupo (I282N) mutation in proline-serine-threonine phosphatase-interacting protein 2 (PSTPIP2) leads to reduced expression of PSTPIP2 that is associated with a macrophage-mediated autoinflammatory disease. Another mutation in PSTPIP2, L98P, termed chronic multifocal osteomyelits (cmo), leads to a disease in mice that resembles chronic recurrent multifocal osteomyelits in humans. The cellular basis of cmo disease was investigated. cmo disease develops independently of lymphocytes and is cured by bone marrow transplantation. Macrophages, mast cells, and osteoclasts from cmo mice fail to express detectable PSTPIP2 protein. Asymptomatic Pstpip2(cmo/cmo) mice have increased circulating levels of macrophage inflammatory protein 1-alpha and interleukin-6, and their macrophages exhibit increased production of these inflammatory mediators, which is normalized by retroviral expression of wild-type PSTPIP2. Spleens of asymptomatic cmo mice contain increased numbers of macrophage precursors, and cmo mice mobilize more macrophage precursors in response to a sterile inflammatory stimulus. Signal transducer and activator of transcription 1 is elevated in cmo splenic macrophages, which also exhibit increased colony-stimulating factor-1-stimulated proliferation and increased extracellular signal-regulated kinase 1/2 phosphorylation. PSTPIP2 overexpression in macrophages leads to the opposite phenotype. Thus, PSTPIP2 deficiency causes both an expansion of macrophage progenitors and increased responsiveness of mature macrophages to activating stimuli, which together prime the organism for exaggerated and sustained responses leading to autoinflammatory disease.

A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing.

Available protocols for stripping antibodies from immunoblots involve the use of sodium dodecyl sulfate (SDS) or low-pH buffers. SDS was shown to remove transferred proteins from membranes, and low-pH buffer was shown to inefficiently strip off antibodies. A solution containing 6M guanidine hydrochloride, 0.2% nondenaturing detergent, and a reducing agent can rapidly strip off tightly bound antibodies from aged polyvinylidene fluoride (PVDF) immunoblots at room temperature without removing significant amounts of transferred protein.

Anthrax lethal toxin triggers the formation of a membrane-associated inflammasome complex in murine macrophages.

Multiple microbial components trigger the formation of an inflammasome complex that contains pathogen-specific nucleotide oligomerization and binding domain (NOD)-like receptors (NLRs), caspase-1, and in some cases the scaffolding protein ASC. The NLR protein Nalp1b has been linked to anthrax lethal toxin (LT)-mediated cytolysis of murine macrophages. Here we demonstrate that in unstimulated J774A.1 macrophages, caspase-1 and Nalp1b are membrane associated and part of approximately 200- and approximately 800-kDa complexes, respectively. LT treatment of these cells resulted in caspase-1 recruitment to the Nalp1b-containing complex, concurrent with processing of cytosolic caspase-1 substrates. We further demonstrated that Nalp1b and caspase-1 are able to interact with each other. Intriguingly, both caspase-1 and Nalp1b were membrane associated, while the caspase-1 substrate interleukin-18 was cytosolic. Caspase-1-associated inflammasome components included, besides Nalp1b, proinflammatory caspase-11 and the caspase-1 substrate alpha-enolase. Asc was not part of the Nalp1b inflammasome in LT-treated macrophages. Taken together, our findings suggest that LT triggers the formation of a membrane-associated inflammasome complex in murine macrophages, resulting in cleavage of cytosolic caspase-1 substrates and cell death.

Removal of detergents from protein digests for mass spectrometry analysis.

Detergents are commonly used for the extraction of hydrophobic proteins and must be removed for sensitive detection of peptides by mass spectrometry. We demonstrate that ethyl acetate is able to extract octylglycoside from a protease digest without loss of peptides or interference with the peptide mass spectral profile. Ethyl acetate extraction was also found to reduce interference by sodium dodecyl sulfate, Nonidet P-40, or Triton X-100 in the mass spectrometry analysis.

CSF-1 receptor structure/function in MacCsf1r-/- macrophages: regulation of proliferation, differentiation, and morphology.

CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.

Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling.

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.

The PCH family member MAYP/PSTPIP2 directly regulates F-actin bundling and enhances filopodia formation and motility in macrophages.

Macrophage actin-associated tyrosine phosphorylated protein (MAYP) belongs to the Pombe Cdc15 homology (PCH) family of proteins involved in the regulation of actin-based functions including cell adhesion and motility. In mouse macrophages, MAYP is tyrosine phosphorylated after activation of the colony-stimulating factor-1 receptor (CSF-1R), which also induces actin reorganization, membrane ruffling, cell spreading, polarization, and migration. Because MAYP associates with F-actin, we investigated the function of MAYP in regulating actin organization in macrophages. Overexpression of MAYP decreased CSF-1-induced membrane ruffling and increased filopodia formation, motility and CSF-1-mediated chemotaxis. The opposite phenotype was observed with reduced expression of MAYP, indicating that MAYP is a negative regulator of CSF-1-induced membrane ruffling and positively regulates formation of filopodia and directional migration. Overexpression of MAYP led to a reduction in total macrophage F-actin content but was associated with increased actin bundling. Consistent with this, purified MAYP bundled F-actin and regulated its turnover in vitro. In addition, MAYP colocalized with cortical and filopodial F-actin in vivo. Because filopodia are postulated to increase directional motility by acting as environmental sensors, the MAYP-stimulated increase in directional movement may be at least partly explained by enhancement of filopodia formation.

The androgen receptor acetylation site regulates cAMP and AKT but not ERK-induced activity.

The androgen receptor (AR) regulates ligand-dependent gene transcription upon binding specific DNA sequences. The AR conveys both trans-activation and trans-repression functions, which together contribute to prostate cellular growth, differentiation, and apoptosis. Like histone H3, the AR is post-translationally modified by both acetylation and phosphorylation. The histone acetyltransferase p300 transactivates the AR and directly acetylates the AR in vitro at a conserved motif. Point mutations of the AR acetylation motif that abrogate acetylation reduce trans-activation by p300 without affecting the trans-repression function of the AR. The current studies assessed the functional relationship between acetylation and phosphorylation of the AR. Herein trans-activation of the AR acetylation site mutants were enhanced by the p42/p44 MAPK pathway but were defective in regulation by protein kinase A (PKA) signaling. PKA inhibition augmented ARwt activity but not AR acetylation mutant gene reporter activity and association at an androgen response element in chromatin immunoprecipitation assays. Mutations of the lysine residues at the AR acetylation site reduced trichostatin A (TSA) responsiveness and ligand-induced phosphorylation of the AR. The AR acetylation site mutant formed ligand-induced phosphorylation-dependent isoforms with distinguishable characteristics from wild type AR as determined with two-dimensional electrophoresis. Conversely, point mutation of a subset of AR phosphorylation sites reduced trichostatin A responsiveness and trans-activation by histone acetyltransferases. Together these studies suggest that acetylation and phosphorylation of the AR are linked events and that the conserved AR lysine motif contributes to a select subset of pathways governing AR activity.

Proteomic approaches to the analysis of early events in colony-stimulating factor-1 signal transduction.

The exposure of cells to growth factors leads to the rapid tyrosine phosphorylation of proteins that play critical roles in initiating cellular responses. These proteins are associated with other nontyrosine-phosphorylated proteins. Together, they represent less than 0.02% of the total cellular protein. To study their functions in growth factor signaling it is necessary to establish their identity, post-translational modifications, and interactions. We have focused on the characterization of this group of proteins during the early response of macrophages to the macrophage growth factor, colony-stimulating factor-1 (CSF-1). We review here the development of approaches to analysis of the rapid CSF-1-induced changes in the CSF-1 receptor tyrosine kinase and phosphotyrosyl signaling complexes. Recent advances in mass spectrometry technology are greatly facilitating the characterization of such complexes. These methods strongly support and enhance genetic approaches that are being used to analyze the function of individual signaling components and pathways.