John M. Aletta

[18] PC12 pheochromocytoma cells: culture, nerve growth factor treatment, and experimental exploitation

Publisher Summary This chapter presents several details regarding its culture and experimental exploitation—in particular, methods for the maintenance and nerve growth factor (NGF) treatment of PC12 cultures as well as for the generation and selection of PC12 cell mutants and for the preparation of a PC12 cell fraction, which is highly enriched in neurites. One of the important advantages of the PC12 cell line is the large amount of homogeneous material it provides for experimental analysis. An example of the use of this asset is the attempt of this chapter to understand the molecular basis of the regional specializations of neurite-bearing cells. To this end, techniques have been devised for obtaining preparations, which are very highly enriched in neurites. A major characteristic of the cells is that they respond to the neuronotrophic agent nerve growth factor (NGF) by slowly shifting from a proliferating chromaffin/pheochromocytoma cell-like phenotype to that of nonproliferating, neurite-bearing sympathetic-like neuron.

Protein methylation: a signal event in post-translational modification

Despite the data discussed here, little is known about the precise mechanisms that regulate protein methylation in eukaryotic cells. Ligand-stimulated regulation of a specific methyltransferase has not been directly demonstrated. In the case of the interferon receptor, histone methylating activity is constitutive and was not altered by treating intact cells for 5 min with interferon β. PRMT1 activity is qualitatively and quantitatively regulated by GST–TIS21 and GST–BTG1, two similar immediate-early gene fusion proteins, but these effects have only been demonstrated in vitro. The evidence for the involvement of protein methylation in differentiation is indirect, since these studies either rely on methylation inhibitors or measure the changes in methylation associated with unidentified, albeit specific, cellular proteins. The effects of methylation on the function of these proteins are also unknown. Thus, further efforts are required to establish the identities of both the enzymes and the substrates involved in regulated protein methylation in any given cellular model. For example, the nonreceptor agonist, elevated glucose, was recently shown to increase the incorporation of base-labile methyl groups (indicative of carboxyl methylation) into anti-CDC42 and anti-Rap1 immunoprecipitates from intact cells[24xKowluru, A. et al. J. Clin. Invest. 1996; 98: 540–555CrossrefSee all References][24]. Additional experiments indicate that carboxyl methylation of G proteins in this system is a requisite step in nutrient-induced insulin secretion. Similar approaches aimed at the identification of specific substrates altered by receptor-mediated events and the identification of the protein methyltransferase and methylesterase involved will further focus the molecular analysis of the role of regulated protein methylation in distinct cellular functions.The abundance of important signaling molecules that are methylated (including PP2A, G proteins, the Ras family of proteins, transcription factors and RNA processing proteins) and recent studies coupling growth factor receptors with protein methylation ensure that the role of protein methylation in cellular signaling events will receive a renewed and deserved interest in the near future.

Methylation regulates the intracellular protein-protein and protein-RNA interactions of FMRP.

FMRP, the fragile X mental retardation protein, is an RNA-binding protein that interacts with ∼4% of fetal brain mRNA. We have recently shown that a methyltransferase (MT) co-translationally methylates FMRP in vitro and that methylation modulates the ability of FMRP to bind mRNA. Here, we recapitulate these in vitro data in vivo, demonstrating that methylation of FMRP affects its ability to bind to FXR1P and regulate the translation of FMRP target mRNAs. Additionally, using double-label fluorescence confocal microscopy, we identified a subpopulation of FMRP-containing small cytoplasmic granules that are distinguishable from larger stress granules. Using the oxidative-stress induced accumulation of abortive pre-initiation complexes as a measure of the association of FMRP with translational components, we have demonstrated that FMRP associates with ribosomes during initiation and, more importantly, that methylation regulates this process by influencing the ratio of FMRP-homodimer-containing mRNPs to FMRP-FXR1P-heterodimer-containing mRNPs. These data suggest a vital role for methylation in normal FMRP functioning.

Nerve growth factor‐mediated increases in protein methylation occur predominantly at type I arginine methylation sites and involve protein arginine methyltransferase 1

Nerve growth factor (NGF)‐specific signal transduction leads to changes in protein methylation during neuronal differentiation of PC12 cells (Cimato et al. [1997] J. Cell Biol. 138:1089–1103). In the present work, we demonstrate that, among NGF‐regulated proteins, arginine methylation is more prevalent than carboxylmethylation. Type I protein arginine methyltransferase (PRMT) activity produces asymmetric dimethylation of the terminal guanidinonitrogen of arginines in substrate proteins, particularly glycine and arginine‐rich (GAR) segments of proteins. Several GAR peptides were used to assay for methyltransferase activity and to compete with endogenous cellular proteins for the PRMT activity in PC12 cell extracts. Peptides derived from fibrillarin and nucleolin, as well as a synthetic GAR peptide containing a repetitive GRG motif, are each extremely effective at blocking in vitro methylation of the NGF‐regulated PC12 cell methylated proteins. Myelin basic protein, a substrate for type II PRMT, selectively inhibits a 45 kDa protein but is a much less effective inhibitor of total methylation at an equimolar concentration. In addition, the fibrillarin‐ and nucleolin‐derived peptides were used to detect elevated PRMT activity in homogenates of NGF‐treated PC12 cells. Finally, immunoprecipitation of PRMT1 from PC12 cells provides the first demonstration of an NGF‐activated methyltransferase and implicates PRMT1 in NGF signal transduction.

Accurate localization and relative quantification of arginine methylation using nanoflow liquid chromatography coupled to electron transfer dissociation and drbitrap mass spectrometry

Protein arginine (Arg) methylation serves an important functional role in eucaryotic cells, and typically occurs in domains consisting of multiple Arg in close proximity. Localization of methylarginine (MA) within Arg-rich domains poses a challenge for mass spectrometry (MS)-based methods; the peptides are highly charged under electrospray ionization (ESI), which limits the number of sequence-informative products produced by collision induced dissociation (CID), and loss of the labile methylation moieties during CID precludes effective fragmentation of the peptide backbone. Here the fragmentation behavior of Arg-rich peptides was investigated comprehensively using electron-transfer dissociation (ETD) and CID for both methylated and unmodified glycine-/Arg-rich peptides (GAR), derived from residues 679–695 of human nucleolin, which contains methylation motifs that are widely-represented in biological systems. ETD produced abundant information for sequencing and MA localization, whereas CID failed to provide credible identification for any available charge state (z=2–4). Nevertheless, CID produced characteristic neutral losses that can be employed to distinguish among different types of MA, as suggested by previous works and confirmed here with product ion scans of high accuracy/resolution by an LTQ/Orbitrap. To analyze MA-peptides in relatively complex mixtures, a method was developed that employs nano-LC coupled to alternating CID/ETD for peptide sequencing and MA localization/characterization, and an Orbitrap for accurate precursor measurement and relative quantification of MA-peptide stoichiometries. As proof of concept, GAR-peptides methylated in vitro by protein arginine N-methyltransferases PRMT1 and PRMT7 were analyzed. It was observed that PRMT1 generated a number of monomethylated (MMA) and asymmetric-dimethylated peptides, while PRMT7 produced predominantly MMA peptides and some symmetric-dimethylated peptides. This approach and the results may advance understanding of the actions of PRMTs and the functional significance of Arg methylation patterns.

Proteomic Analysis Reveals Diverse Classes of Arginine Methylproteins in Mitochondria of Trypanosomes

Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Additionally, trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric analysis of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatography separation, and high-resolution mass spectrometry analysis with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metabolism, transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive analysis of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.

Activation of a Calcium-Calmodulin-dependent Protein Kinase I Cascade in PC12 Cells

It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7.3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by ∼80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.

Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation

UNLABELLED Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of Trypanosoma brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. BIOLOGICAL SIGNIFICANCE T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varying milieus. One mechanism by which all cells respond to changing environments is through posttranslational modification of proteins. Arginine methylation is one such modification that can dramatically impact protein-protein and protein-nucleic acid interactions and subcellular localization of proteins. To define the breadth of arginine methylation in trypanosomes and identify target proteins, we performed a global proteomic analysis of arginine methylated proteins in insect stage T. brucei. We identified 1332 methylarginines in 676 proteins, generating the largest compilation of methylarginine containing proteins in any organism to date. Numerous arginine methylated proteins function in RNA and DNA related processes, suggesting this modification can impact T. brucei genome integrity and gene regulation at numerous points. Other processes that appear to be strongly influenced by arginine methylation are intracellular protein trafficking, signaling, protein folding and degradation, and flagellar function. The widespread nature of arginine methylation in trypanosomes highlights its potential to greatly affect parasite biology and pathogenesis.

Arginine methylation of a mitochondrial guide RNA binding protein from Trypanosoma brucei.

RBP16 is a mitochondrial Y-box protein from the parasitic protozoan Trypanosoma brucei that binds guide RNAs and ribosomal RNAs. It is comprised of an N-terminal cold-shock domain and a C-terminal domain rich in glycine and arginine residues, resembling the RGG RNA-binding motif. Arginine residues found within RGG domains are frequently asymmetrically dimethylated by a class of enzymes termed protein arginine methyltransferases (PRMTs). As Arg-93 of RBP16 exists in the context of a preferred sequence for asymmetric arginine dimethylation (G/FGGRGGG/F), we investigated whether modified arginines are present in native RBP16 by MALDI-TOF and post-source decay analyses. These analyses confirmed that Arg-93 is dimethylated. In addition, Arg-78 exists as an unmodified or as a monomethylated derivative, and Arg-85 is present in forms corresponding to the unmodified, di-, and tri-methylated state. While Arg-93 is apparently constitutively dimethylated, the methylation of Arg-78 and Arg-85 is mutually exclusive. Furthermore, whole cell extracts from procyclic form T. brucei are able to methylate bacterially expressed RBP16 (rRBP16), as well as endogenous proteins, in the presence of S-adenosyl-L-[methyl-3H]methionine. While assays of mitochondrial extracts suggest a small amount of PRMT may also be present in this subcellular compartment, the majority of trypanosome PRMT activity is extramitochondrial. We show that rRBP16 is methylated in trypanosome extracts through the action of a type I methyltransferase as well as serving as a substrate for heterologous mammalian type I PRMTs. In addition, we demonstrate the presence of type II PRMT activity in trypanosome cell extracts. These results suggest that protein arginine methylation is a common posttranslational modification in trypanosomes, and that it may regulate the function of RBP16.

A novel nuclear FGF Receptor‐1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells

FGF Receptor‐1 (FGFR1), a membrane‐targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal‐like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA‐induced differentiation while full‐length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co‐associate with 5′‐Fluorouridine‐labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf‐2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter‐gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation. J. Cell. Biochem. 113: 2920–2936, 2012.