The role of atypical protein kinase C in CSF-1-dependent Erk activation and proliferation in myeloid progenitors and macrophages.

Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States, Mol. Cell. Biol.18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.

Gab2 promotes colony-stimulating factor 1-regulated macrophage expansion via alternate effectors at different stages of development.

Colony-stimulating factor 1 (CSF-1) receptor (CSF-1R, or macrophage CSF receptor [M-CSFR]) is the primary regulator of the proliferation, survival, and differentiation of mononuclear phagocytes (MNPs), but the critical CSF-1 signals for these functions are unclear. The scaffold protein Gab2 is a major tyrosyl phosphoprotein in the CSF-1R signaling network. Here we demonstrate that Gab2 deficiency results in profoundly defective expansion of CSF-1R-dependent MNP progenitors in the bone marrow, through decreased proliferation and survival. Reconstitution and phospho-flow studies show that downstream of CSF-1R, Gab2 uses phosphatidylinositol 3-kinase (PI3K)-Akt and extracellular signal-regulated kinase (Erk) to regulate MNP progenitor expansion. Unexpectedly, Gab2 ablation enhances Jun N-terminal protein kinase 1 (JNK1) phosphorylation in differentiated MNPs but reduces their proliferation; inhibition of JNK signaling or reduction of JNK1 levels restores proliferation. MNP recruitment to inflammatory sites and the corresponding bone marrow response is strongly impaired in Gab2-deficient mice. Our data provide genetic and biochemical evidence that CSF-1R, through Gab2, utilizes different effectors at different stages of MNP development to promote their expansion.

Measurement of dissociation rate of biomolecular complexes using CE.

Fluorescence anisotropy (FA), non-equilibrium CE of equilibrium mixtures (NECEEM) and high-speed CE were evaluated for measuring dissociation kinetics of peptide-protein binding systems. Fyn-SH3-SH2, a protein construct consisting of the src homology 2 (SH2) and 3 (SH3) domain of the protein Fyn, and a fluorescein-labeled phosphopeptide were used as a model system. All three methods gave comparable half-life of approximately 53 s for Fyn-SH3-SH2:peptide complex. Achieving satisfactory results by NECEEM required columns over 30 cm long. When using Fyn-SH2-SH3 tagged with glutathione S-transferase (GST) as the binding protein, both FA and NECEEM assays gave evidence of two complexes forming with the peptide, yet neither method allowed accurate measurement of dissociation rates for both complexes because of a lack of resolution. High-speed CE, with a 7 s separation time, enabled separation of both complexes and allowed determination of dissociation rate of both complexes independently. The two complexes had half-lives of 22.0+/-2.7 and 58.8+/-6.1 s, respectively. Concentration studies revealed that the GST-Fyn-SH3-SH2 protein formed a dimer so that complexes had binding ratios of 2:1 (protein-to-peptide ratio) and 2:2. Our results demonstrate that although all methods are suitable for 1:1 binding systems, high-speed CE is unique in allowing multiple complexes to be resolved simultaneously. This property allows determination of binding kinetics of complicated systems and makes the technique useful for discovering novel affinity interactions.

Integration of genome and chromatin structure with gene expression profiles to predict c-MYC recognition site binding and function.

The MYC genes encode nuclear sequence specific-binding DNA-binding proteins that are pleiotropic regulators of cellular function, and the c-MYC proto-oncogene is deregulated and/or mutated in most human cancers. Experimental studies of MYC binding to the genome are not fully consistent. While many c-MYC recognition sites can be identified in c-MYC responsive genes, other motif matches-even experimentally confirmed sites-are associated with genes showing no c-MYC response. We have developed a computational model that integrates multiple sources of evidence to predict which genes will bind and be regulated by MYC in vivo. First, a Bayesian network classifier is used to predict those c-MYC recognition sites that are most likely to exhibit high-occupancy binding in chromatin immunoprecipitation studies. This classifier incorporates genomic sequence, experimentally determined genomic chromatin acetylation islands, and predicted methylation status from a computational model estimating the likelihood of genomic DNA methylation. We find that the predictions from this classifier are also applicable to other transcription factors, such as cAMP-response element-binding protein, whose binding sites are sensitive to DNA methylation. Second, the MYC binding probability is combined with the gene expression profile data from nine independent microarray datasets in multiple tissues. Finally, we may consider gene function annotations in Gene Ontology to predict the c-MYC targets. We assess the performance of our prediction results by comparing them with the c-myc targets identified in the biomedical literature. In total, we predict 460 likely c-MYC target genes in the human genome, of which 67 have been reported to be both bound and regulated by MYC, 68 are bound by MYC, and another 80 are MYC-regulated. The approach thus successfully identifies many known c-MYC targets and suggests many novel sites. Our findings suggest that to identify c-MYC genomic targets, integration of different data sources helps to improve the accuracy.

Evolutionary-conserved gene expression response profiles across mammalian tissues.

Gene expression responses are complex and frequently involve the actions of many genes to effect coordinated patterns. We hypothesized these coordinated responses are evolutionarily conserved and used a comparison of human and mouse gene expression profiles to identify the most prominent conserved features across a set of normal mammalian tissues. Based on data from multiple studies across multiple tissues in human and mouse, 13 gene expression modes across multiple tissues were identified in each of these species using principal component analysis. Strikingly, 1-to-1 pairing of human and mouse modes was observed in 12 out of 13 modes obtained from the two species independently. These paired modes define evolutionarily conserved gene expression response modes (CGEMs). Notably, in this study we were able to extract biological responses that are not overwhelmed by laboratory-to-laboratory or species-to-species variation. Of the variation in our gene expression dataset, 84% can be explained using these CGEMs. Functional annotation was performed using Gene Ontology, pathway, and transcription factor binding site over representation. Our conclusion is that we found an unbiased way of obtaining conserved gene response modes that accounts for a considerable portion of gene expression variation in a given dataset, as well as validates the conservation of major gene expression response modes across the mammals.

Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis.

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-Bbeta, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bbeta and Fyn complex barely affected. IC(50) of both selective and nonselective inhibitors were determined and compared for different proteins. The IC(50) of the nonselective inhibitor was 49 +/- 9, 323 +/- 42, and 228 +/- 19 microM (n = 3) for Src, SH2-Bbeta, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions.

A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid-induced neuronal differentiation.

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase-AKT.

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation.

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.