Identification of the Chicken MARCKS Phosphorylation Site Specific

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Identification of the Chicken MARCKS Phosphorylation Site Specific for Differentiating Neurons as Ser 25 Using a Monoclonal Antibody and Mass Spectrometry Flavio R. Zolessi,† Rosario Dura´ n,‡ Ulla Engstro1 m,§ Carlos Cerven ˜ ansky,‡ Ulf Hellman,§ and ,† Cristina Arruti* Laboratorio de Cultivo de Tejidos, Seccio´n Biologı´a Celular, DBCM, Facultad de Ciencias, Universidad de la Repu ´ blica, Uruguay, Laboratorio de Bioquı´mica Analı´tica, IIBCE y Facultad de Ciencias, Universidad de la Repu ´ blica, Uruguay, and Protein Structure Group, Ludwig Institute for Cancer Research, Uppsala, Sweden Received August 20, 2003

MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and prolinedirected kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development. Keywords: protein phosphorylation • phosphorylation-specific antibody • proline-directed kinases • Cdk • neural differentiation • mass spectrometry

Introduction The regulation of cellular processes largely depends on gene expression and post-translational modifications of proteins. Of these, phosphorylation is known as a versatile way of changing protein activity, either grossly (activating or inactivating proteins) or finely modulating their properties.1 Then, the elucidation of the network of intracellular interactions, that may respond to extrinsic or intrinsic signals, and that determine the status of protein phosphorylation in a cell (the phosphoproteome2), appears as a pivotal step for the understanding of cell function and behavior. But, the overwhelming complexity of these molecular interactions has precluded their complete understanding in the past. Maybe a possible initial clue for this understanding comes from the comprehensive study of proteins that play roles as “connecting devices” between different intracellular signaling pathways. The “Myristoylated AlanineRich C Kinase Substrate” (MARCKS) is one of such proteins.3,4 MARCKS main function in cells seems to be related to actin dynamics regulation, either directly by a microfilament crosslinking activity5 or indirectly through the modulation of the lipid second messenger phosphatidyl-inositol(4,5)bisphosphate (PIP2) at plasma membrane rafts.6 Both activities (actin filament cross-linking and PIP2 binding) are concentrated on a basic central “effector domain” (ED) and are inhibited by protein kinase C (PKC) phosphorylation in up to three serine residues, located in the ED, or by calcium-bound calmodulin (Ca-CaM) binding, also at the ED.5,7 Membrane binding depends on the cooperation between the electrostatic interactions between the * To whom correspondence should be addressed. Tel: (5982) 525-8618 ext 144. Fax: (5982) 525-8629. E-mail: [email protected] † Laboratorio de Cultivo de Tejidos, Seccio´n Biologı´a Celular, DBCM, Facultad de Ciencias, Universidad de la Repu ´ blica. ‡ Laboratorio de Bioquı´mica Analı´tica, IIBCE y Facultad de Ciencias, Universidad de la Repu ´ blica. § Protein Structure Group, Ludwig Institute for Cancer Research.

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Published on Web 11/04/2003

positively charged ED and negatively charged lipids (mainly PIP28) and the insertion of a lipid moiety (myristate) covalently bound to MARCKS amino-terminus.9,10 Consequently, this interaction may also be regulated by a cycle of myristoylation/ demyristoylation.11 Finally, several other in vivo phosphorylation sites have been found in MARCKS sequence outside the ED, all of them for proline-directed kinases (PDKs).12,13 Although the exact function of these phosphorylations has not been established, they might modulate MARCKS function in different ways. For instance, it has been shown that phosphorylation by Erk1/2 occurs in vivo in cultured neurons, and that it may inhibit actin cross-linking and Ca-CaM binding activities,14 whereas there are evidences suggesting that phosphorylation by cyclin-dependent kinases (Cdks) causes a contrary effect, augmenting MARCKS affinity for Ca-CaM.15 Unfortunately, direct evidence indicating which sites are phosphorylated by these kinases in vivo is presently lacking. We have previously described the generation of a monoclonal antibody, by immunization of mice with chick embryo neural retina, that recognizes MARCKS only in neuronal cells.16 But MARCKS is a ubiquitous protein,17 so we investigated the possibility that mAb 3C3 recognizes a modified isoform of the protein. We then demonstrated that this antibody only binds to a phosphorylated MARCKS isoform, already present in neurons at early stages of their differentiation and then preserved in a sustained manner throughout the histogenetic period of the neural retina and other organs of the central nervous system.18 Interestingly, knock-out mice lacking MARCKS expression show a phenotype characterized by defects in different events of neural development, including neural tube closure, brain commisures formation, and cortical and retinal histogenesis.19 Thus, the identification of a neuronal-specific phosphorylation could help in the understanding of MARCKS specific function in neural development, and a monoclonal 10.1021/pr034066f CCC: $27.50

 2004 American Chemical Society

MARCKS Phosphorylation at Ser 25 in Neurons

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antibody recognizing such a modification would become an essential molecular tool that could be used to achieve this task. In the present paper, we describe the identification of the residue that must be phosphorylated for mAb 3C3 binding as Ser 25, a site located in a consensus sequence for Cdks, and the partial elucidation of the epitope for this antibody on MARCKS sequence. In addition, we found the existence of other putatively in vivo phosphorylated proline-directed kinases sites in MARCKS in chick embryo neural tissues. In conjunction, these and our previous findings suggest a complex regulation of MARCKS by phosphorylation during neural development, and open a new path for the investigation of its role in this and other cellular processes.

Materials and Methods Animals. Fertilized hen eggs were kindly donated by Prof. Dr. H. Trenchi, Facultad de Veterinaria, Universidad de la Repu ´ blica, and incubated in our laboratory at 37 °C in humidified atmosphere until the desired stage. Retinas or brains were dissected in ice-cold PBS and immediately used or stored frozen at -20 °C. Antibodies and Immunoblotting. We used two primary antibodies recognizing MARCKS: mAb 3C316 and MCt (rabbit polyclonal anti-carboxy-terminal MARCKS20). Electrophoretically separated proteins (10% polyacrylamide gel) were electrotransferred, or protein/peptides solutions were dotted onto nitrocellulose membrane (Sigma, St. Louis, MO) and processed for immunodetection with the described primary antibodies. Secondary antibodies were peroxidase conjugated (anti-rabbit IgG: Pierce, Rockford, IL, USA; anti-mouse IgG: Sigma, St. Louis, MO). Immunoreaction was revealed by chemiluminescence using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Sequential immunodetection on the same blot was as previously described;18 briefly, once the first immunodetection was performed, membrane-bound peroxidase was inactivated with 0.1% sodium azide followed by the second immunoreaction. For Western-blot analysis of MARCKS incomplete tryptic digestion products, we incubated a post-boiling supernatant of chick embryo neural retinas with 50 µg/mL trypsin for 10 min, at 37 °C. Dephosphorylation of protein/peptides dotted on nitrocellulose was as described for Western-blots.18 Protein Purification, Mass Spectrometry and Synthetic Peptides. MARCKS was purified from chick embryo brains (E12-14) by immunoaffinity chromatography with mAb 3C3 as previously described.16 An improvement introduced in some purification protocols used in this work included boiling the brain homogenate for 10 min prior to the affinity purification step, a procedure that lowered the total quantity of proteins and inhibited most of the endogenous phosphatase and protease activities in the supernatant (where we recovered MARCKS). The purified protein was analyzed by mass spectrometry by applying the protein to the MALDI target, using the double layer method, with sinapinic acid (SA) as the matrix. MS analyses were made either in an Autoflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany), or in a Voyager DE PRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA), both in linear mode. The measurements were externally calibrated using enolase as a standard. We used two approaches to map mAb 3C3 epitope on MARCKS sequence. In the first one, we generated tryptic fragments from 20 µg immunopurified MARCKS by treating

Figure 1. Procedure followed for mapping mAb 3C3 epitope on MARCKS. Schematic representation showing the main steps in the procedure (see Materials and Methods for details).

with 0.2 µg modified trypsin, sequencing grade (Promega, Madison, WI) in a reaction volume of 100 µL, for 2 h at 30 °C. Peptides were then separated by a RP-HPLC in a C18 column (150 × 2.1 mm; Grace Vydac, Anaheim, CA), and eluted with a linear acetonitrile gradient (0-40%) in a LKB system. We analyzed all the fractions by dot-blot (1 µL/fraction) with mAb 3C3, and the positive ones were then applied to the MALDI target by the dried droplet method, with a saturated solution of R-cyano 4-hydroxy-cinnamic acid (R-CCA) in 0.2% trifluoroacetic acid (TFA). Peptide masses were then analyzed in a Voyager DE PRO MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA), in linear mode. For peptides we also used a external calibration, with different protein standards. The second procedure followed to map the epitope was a modification of the one described by Peter and Tomer21 and is schematically represented in Figure 1. Fresh or frozen retinas were homogenized in PBS containing 0.2% Triton X-100, 2 mM PMSF, 1 mM EDTA, 1 mM EGTA, 0.01 mg/mL aprotinin, 0.16 mg/mL benzamidine, 0.02 mg/mL iodoacetamide at 0 °C. After boiling for 10 min and centrifugation, the supernatant was incubated in batch with mAb 3C3 covalently bound to Sepharose beads and washed thoroughly with the homogenization buffer, followed by PBS alone and last with 50 mM Tris, pH 8.0. Modified trypsin was added to a final concentration of 1 µg/ mL and the mixture incubated for different times at 30 °C. After washing with PBS and water, beads were either mixed 1:1 (v/ v) with a saturated solution of R-CCA and directly dried on the MALDI target, or alternatively eluted with 1% TFA, and analyzed in the Voyager DE PRO mass spectrometer in linear mode. The eluate was directly analyzed by MS as described, or it was reincubated with mAb 3C3-Sepharose after eliminating TFA. These latter complexes were then processed for MS as described. For PSD fragmentation analyses, the eluate from the trypsin-digested Sepharose beads was used, taking the 3156 Da peptide as the precursor ion. Two peptides, like those described in Figure 6, were synthesized following the Fmoc (9-fluorenylmethoxycarbonyl) chemistry with Fmoc-Amide Resin, resulting in C-terminally amidated peptides. Purity of peptides was tested by RP-HPLC using C18 column and correct molecular weight was confirmed by MALDI-TOF MS. Journal of Proteome Research • Vol. 3, No. 1, 2004 85

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Figure 2. Initial characterization of MARCKS recognized by mAb 3C3. A, MALDI-TOF MS analysis of MARCKS immunopurified with mAb 3C3. B, Western-blot of a neural retina lysate mildly digested with trypsin, using mAb 3C3 and the polyclonal antibody directed to the carboxy-terminus of MARCKS (MCt).

Results and Discussion Initial Characterization of mAb 3C3-Immunoreactive MARCKS by Mass Spectrometry and Western-Blot. Analysis of immunoaffinity purified MARCKS from chick embryonic brain (E12-13) by MALDI-TOF MS, gave a main signal centered at m/z 27.8 kDa (Figure 2A). This is the first direct mass measure of chicken protein given that up to now the known value of 27.6 kDa was predicted from translation of nucleotide sequence.22 In addition, we observed two more peaks at m/z 13.9 kDa and 55.6 kDa (Figure 2A). The latter could represent a dimer of the protein, either natural, or generated during ionization, whereas the smallest one most probably represents the doubly charged ion. The possible existence of MARCKS dimers was already suggested after observation by electron microscopy of purified bovine protein.5 As it has been previously described, MARCKS migrated in SDS-PAGE like a protein with a higher molecular weight,3 and this 71 kDa band was recognized both by our mAb 3C3 and by a polyclonal antibody directed to the carboxy-terminus of the protein (MCt)(Figure 2B). To roughly locate the mAb 3C3 epitope on MARCKS, we performed limited tryptic digestion of the protein and then compared the immunoreactivity of the products with both antibodies, using a sequential Western-blot analysis.18 MCt recognized a band with a Mr of 35 kDa, that can be defined as the C-terminal half of the protein, whereas mAb 3C3 did not bind to this band, but recognized instead a 39 kDa one (Figure 2B). Taking into account that the part of the protein most sensitive to tryptic digestion is its central ED region, containing a cluster of 13 lysines,3 this experiment strongly suggested that mAb 3C3 epitope is located in the N-terminal half. Immunoreactivity and Mass Analysis of RP-HPLC-Separated MARCKS Proteolytic Peptides. In addition to the ED 86

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Figure 3. Results obtained from the application of the first method for epitope mapping. A, RP-HPLC profile of the resulting peptides after partial digestion with trypsin of immunopurified MARCKS. B, dot-blot analysis with mAb 3C3 of the RP-HPLC fractions shown in A. C, MALDI-TOF MS analysis of the peptide mixture and the three mAb 3C3-positive fractions from the RPHPLC. D, overlapping MARCKS sequences that, if phosphorylated, could correspond to two of the mass peaks observed in the three mAb 3C3-positive fractions. 11-38 in F13 contains only one putative phosphorylation site, Ser 25, whereas 11-80 in F19/ F20 contains Ser 25 and Ser 44 as putative phosphorylation sites. E, MARCKS sequence corresponding to the mass peaks 6115, 6195, and 6275 Da (found in fractions 19 and 20), assuming that the first one is not phosphorylated, the second has one, and the third two, phosphate groups. The three putative phosphorylation sites (for PDKs) are marked with arrowheads.

serines (phosphorylatable by PKC), the N-terminal moiety of the protein bears most of the putative proline-directed kinase sites (four SP and two TP motifs), consequently, to define the phosphorylated residue responsible for mAb 3C3 binding, a more accurate approach was needed. Therefore, we tried to separate tryptic peptides obtained from immunopurified chick MARCKS by RP-HPLC, and then to identify mAb 3C3-positive fractions by MALDI-TOF MS analysis. As we observed that a complete digestion with trypsin or V8 Glu-C protease caused a loss of mAb 3C3 immunoreactivity (not shown), we performed controlled, incomplete digestion with trypsin. Results from a representative experiment are summarized in Figure 3. The digestion yielded a complex peptide mixture that was separated

MARCKS Phosphorylation at Ser 25 in Neurons

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Figure 4. Results obtained from the application of the second method for epitope mapping, I. In this case, a complex of MARCKS with mAb 3C3-Sepharose was tryptically digested for different times. MALDI-TOF mass spectra from the direct analysis of mAb 3C3Sepharose beads containing MARCKS fragments are shown. Control, mAb 3C3-Sepharose beads alone. *, mass peak corresponding to the double-charged peptide of 10742 Da.

as several overlapping peaks by RP-HPLC (Figure 3A), although this complexity was not evident in mass spectra of the mixture (Figure 3C). When we analyzed these RP-HPLC fractions by dotblot with mAb 3C3, we found only three positive ones, which were processed for MALDI-TOF MS (Figure 3B and C). These spectra contained more than one mass peak, making the identification of the putative epitopic region difficult. Nevertheless, we found that at least two of the mass peaks in the three fractions (2787 Da in F13 and 6842 Da in F19 and F20) overlapped in a region containing only one possible PDKs phosphorylation site: serine 25 (Figure 3D). In addition, fractions 19 and 20 contained different quantities of three mass peaks that differed in 80 Da: 6115, 6195 and 6275 Da (Figure 3C and E), strongly suggesting the existence of differently phosphorylated forms of the same peptide. In fact, these results suggested the natural occurrence of doubly-, singly-, and nonphosphorylated forms of the MARCKS region between residues 39 and 101 since they were differentially separated in two RP-HPLC fractions, where, as expected, the doubly phosphorylated peptide eluted before the singly- and nonphosphorylated ones. Three proline-directed kinase sites are located in this region of the protein: Ser 44, Ser 92, and Ser 99 (Figure 3E). Ser 44 is also found in the putative MARCKS sequence for the 6842 Da peak, that contains Ser 25 (Figure 3D). The three corresponding sites exist in the bovine protein sequence, and were found phosphorylated in calf brain by Taniguchi et al.12

When we compared the sequences from different species, we found the following: (a) Ser 44 is located in a sequence that does not correspond to any known phosphorylation consensus, but it is highly conserved; (b) Ser 92 is in a consensus sequence for Erk MAP kinase (P-X-S/T-P23), but it is poorly conserved (the SP motif is lacking in the human protein); (c) Ser 99 is located in a sequence that could be phosphorylated by Cdks with a low efficiency,24 and it is also relatively well conserved. In addition, chick MARCKS Ser 92 corresponds to mouse MARCKS Ser 113, that can be phosphorylated by Erk2 in vitro.25 This complexity in MARCKS phosphorylation pattern, particularly in the nervous tissue, is coherent with our finding of several (8-9) isoelectric point variants of the protein obtained from chick embryo retina.18 Mass Analysis of Tryptic MARCKS Peptides Protected by mAb 3C3. A stronger indication that Ser 25 was the phosphorylated site recognized by mAb 3C3, came from the application of a procedure involving the tryptic digestion of MARCKS protected by the antibody (as summarized in Figure 1). Several approaches to map epitope regions using mass spectrometry, but differing in the method of sample preparation, have been described in the past;26-28 the main advantage of our method was that the antigen (MARCKS) was bound to the immobilized antibody directly from a complex protein mixture. In addition, the whole procedure took just a few hours, and the results were consistently reproduced. We first analyzed by MALDI-TOF MS Journal of Proteome Research • Vol. 3, No. 1, 2004 87

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Figure 5. Results obtained from the application of the second method for epitope mapping, II. A, the tryptic fragments obtained by digesting the complex of MARCKS-mAb 3C3-Sepharose for 2 h were eluted and re-incubated with fresh mAb 3C3-Sepharose. Here, we show the direct analysis of this second complex by MALDI-TOF MS, and, in the insert, the remaining MS signal in the eluate after incubation with mAb 3C3-Sepharose (“flowthrough”). *, mass peaks corresponding to double-charged peptides of 3156, 9250, and 10 740 Da. B, correspondence of MARCKS sequences with some of the mass peaks observed in different experiments: all of them overlapping in a small region containing Ser 25 as the only putative phosphorylation site.

the peptides remaining bound to mAb 3C3 after different incubation times with trypsin (Figure 4). As expected, bigger fragments were found after shorter digestion times, and no mass peaks could be detected after a long (5 h) digestion (Figure 4). At a time of 2 h of digestion, we found the smallest peptides, with a relatively higher signal. These peptides were eluted and re-incubated with fresh mAb 3C3-Sepharose gel. When we analyzed this gel directly by MALDI-TOF MS, we observed the same mass peaks that were present in the original digested complex, but with a higher signal-to-noise ratio (Figure 5A). In addition, other peptides not seen in the first complex or the eluate became now evident (Figure 5A). The resulting flow-through fraction after this incubation was almost devoid of any detectable MARCKS peptides (Figure 5A). Nearly all of the bound peptides could be identified as putative mono-phosphorylated MARCKS fragments, and, moreover, all of them overlapped in the region containing Ser 25 (Figure 5B). The two peptides giving the highest signal, at m/z 2785 and 3156, corresponded to MARCKS residues 11-38 and 7-38 respectively, both containing Ser 25 as the only residue included in a SP motif (Figure 5B). The 3156 Da fragment was selected to perform PSD fragmentation analysis, which confirmed that this peptide was serine- or threonine-phosphorylated, and was helpful to assess the sequence of the fragment (data not shown). Immunoreactivity of Synthetic MARCKS Peptides Bearing Phosphorylated Ser 25. Taking into account the results exposed above, and our previous observations showing that MARCKS is indeed phosphorylated in vivo at Ser 25 in chick embryo neural tissues,16 we decided to finally confirm our 88

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Figure 6. MAb 3C3 immunoreactivity of synthetic peptides containing MARCKS sequence 16-35. A, the synthetic peptides containing Ser 25 phosphorylated (S25p) or unphosphorylated (S25up) and their analysis by dot-blot with mAb 3C3. B, competition assay on Western-blot analysis of chick embryo neural retina, where the membrane was co-incubated with mAb 3C3 and different concentrations of S25p and S25up. Western-blot was performed on thin strips, whose widths were not identical, explaining some of the apparent differences in signal intensity. Control, conventional Western-blot with mAb 3C3 alone. C, dephosphorylation assay on dotted MARCKS protein and S25p peptide.

supposition by synthesizing two peptides containing the MARCKS sequence between residues 16 and 35, one of them phosphorylated at Ser 25 (Figure 6A). In dot-blot assays, mAb 3C3 only recognized the phosphorylated peptide (S25p), even when very low peptide quantities, close to the detection limit of the technique (picomoles) were used (Figure 6A). This result was confirmed by incubating both peptides with mAb 3C3 covalently bound to Sepharose and analyzing the washed beads by MALDI-TOF MS (not shown). S25p, but not S25up (the unphosphorylated peptide), competed with MARCKS for mAb 3C3 binding when co-incubated with the antibody in a Western-blot assay, in the correct concentration ratio (Figure 6B). Moreover, dotted S25p immunoreactivity with mAb 3C3 diminished as a function of incubation time with calf intestine alkaline phosphatase, as occurred with the whole protein (Figure 6C). Data presented here show that the epitope recognized by mAb 3C3 on MARCKS protein includes Ser 25 and that this residue must be phosphorylated for antibody binding. Using synthetic peptides, we have also restricted the epitopic region of this antibody to a sequence of twenty amino acids (from residue 16 to 35). We found evidences indicating that the integrity of the peptide bond between the lysine in position 28 and the alanine 29 may also be necessary because complete digestion with trypsin results in the absolute loss of immunoreactivity, and we never found mAb 3C3-positive fractions containing a peptide cleaved in this position. By now, we cannot ascertain if the epitope is mostly dependent on sequence, or if some contribution by structure is relevant. The observation that mAb 3C3 was able to bind small (20-30 amino acids) peptides containing the epitope suggests a low, if any, dependence on three-dimensional structure. This suggestion is also supported by our previous data showing mAb 3C3 binding to MARCKS in different conditions.16,29 In addition, most of the studies conducted to date in search of the MARCKS

MARCKS Phosphorylation at Ser 25 in Neurons

secondary or tertiary structure, have shown that this protein is mostly in an extended, random-coil conformation.5,30-33 Ser 25 has been previously shown to be phosphorylated in MARCKS purified from chick embryo brain16 and the corresponding residue (Ser 26) was also found phosphorylated in calf brain.12 But, these early works did not show that it was specific for neurons (not even that it was specific for nervous system). The present data, when taken together with our previous ones,16,18 show a tight developmental regulation of MARCKS phosphorylation at Ser 25. In particular, we have found the restriction of mAb 3C3 immunoreactivity to differentiating neurons in the chick,16 as well as that this immunoreactivity (revealing the presence of Ser 25 phosphorylatedMARCKS) appears a short time after retinal ganglion cells last mitosis,18 while total MARCKS immunoreactivity was ubiquitous and found even before the onset of neurodifferentiation.18,34 Ser 25 is located in a consensus sequence for Cdks (S/T-P-X-K/H/R23,24). More specifically, this is the preferred phosphorylation sequence for Cdk1, Cdk2, and Cdk5. Of these kinases, the former two are involved in cell cycle regulation, while the third one is only active in neurons.35,36 The site in the mouse MARCKS sequence corresponding to chick Ser 25 has been shown to be phosphorylated by Cdk2 in vitro with a high efficiency,37 and unidentified MARCKS serines and threonines can be phosphorylated in vitro by Cdk1 and Cdk5.15 Hence, the most probable in vivo kinase for Ser 25 seems to be Cdk5, the neuronal Cdk. Finally, our previous results suggest that the phosphorylation at Ser 25 does not directly affect MARCKS association to the plasma membrane,16,18 where it is supposed to be in an active state.6 It is tempting to speculate that this phosphorylation plays a very different role to that catalyzed by PKC, which inactivates the protein by an “electrostatic switch” mechanism, in an acutely regulated fashion.7 Phosphorylation at Ser 25 could, instead, be finely modulatory and regulated more slowly, what would allow to sustain this modification for the duration of a long process like neuronal cell differentiation.

Acknowledgment. This work was supported in part by PEDECIBA and CSIC, Universidad de la Repu ´ blica, Uruguay. We thank Dr. H. Trenchi for supplying fertilized hen eggs and Dr. Pico Caroni for the anti-MARCKS policlonal antibody (MCt). References (1) Johnson, L. N.; Lewis, R. J. Structural basis for control by phosphorylation. Chem. Rev. 2001, 101, 2209-2242. (2) Jensen, O. N. Modification-specific proteomics: systematic strategies for analysing posttranslationally modified proteins. Proteomics: A Trends Guide; Elsevier Science Ltd.: Holland, 2000; pp. 36-42. (3) Blackshear, P. J. The MARCKS family of cellular protein kinase C substrates. J. Biol. Chem. 1993, 268, 1501-1504. (4) Arbuzova, A.; Schmitz, A. A.; Verge`res, G. Cross-talk unfolded: MARCKS proteins. Biochem. J. 2002, 362, 1-12. (5) Hartwig, J. H.; Thelen, M.; Rosen, A.; Janmey, P. A.; Nairn, A. C.; Aderem, A. MARCKS is an actin filament cross-linking protein regulated by protein kinase C and calcium-calmodulin. Nature 1992, 356, 618-622. (6) Laux, T.; Fukami, K.; Thelen, M.; Golub, T.; Frey, D.; Caroni, P. GAP43, MARCKS, and CAP23 modulate PI(4, 5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. J. Cell Biol. 2000, 149, 1455-1472. (7) McLaughlin, S.; Aderem, A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 1995, 20, 272-276. (8) McLaughlin, S.; Wang, J.; Gambhir, A.; Murray, D. PIP(2) and proteins: interactions, organization, and information flow. Annu. Rev. Biophys. Biomol. Struct. 2002, 31, 151-175.

research articles (9) George, D. J.; Blackshear, P. J. Membrane association of the myristoylated alanine-rich C kinase substrate (MARCKS) protein appears to involve myristate-dependent binding in the absence of a myristoyl protein receptor. J. Biol. Chem. 1992, 267, 24 87924 885. (10) Taniguchi, H.; Manenti, S. Interaction of myristoylated alaninerich protein kinase C substrate (MARCKS) with membrane phospholipids. J. Biol. Chem. 1993, 268, 9960-9963. (11) Manenti, S.; Sorokine, O.; Van Dorsselaer, A.; Taniguchi, H. Isolation of the nonmyristoylated form of a major substrate of protein kinase C (MARCKS) from bovine brain. J. Biol. Chem. 1993, 268, 6878-6881. (12) Taniguchi, H.; Manenti, S.; Suzuki, M.; Titani, K. Myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, is an in vivo substrate of proline-directed protein kinase(s). A mass spectroscopic analysis of the post-translational modifications. J. Biol. Chem. 1994, 269, 18 299-18 302. (13) Yamauchi, E.; Kiyonami, R.; Kanai, M.; Taniguchi, H. The Cterminal conserved domain of MARCKS is phosphorylated in vivo by proline-directed protein kinase. Application of ion trap mass spectrometry to the determination of protein phosphorylation sites. J. Biol. Chem. 1998, 273, 4367-4371. (14) Ohmitsu, M.; Fukunaga, K.; Yamamoto, H.; Miyamoto, E. Phosphorylation of myristoylated alanine-rich protein kinase C substrate by mitogen-activated protein kinase in cultured rat hippocampal neurons following stimulation of glutamate receptors. J. Biol. Chem. 1999, 274, 408-417. (15) Yamamoto, H.; Arakane, F.; Ono, T.; Tashima, K.; Okumura, E.; Yamada, K.; Hisanaga, S.; Fukunaga, K.; Kishimoto, T.; Miyamoto, E. Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) by proline-directed protein kinases and its dephosphorylation. J. Neurochem. 1995, 65, 802-809. (16) Zolessi, F. R.; Baz, A.; Hellman, U.; Arruti, C. Characterization of MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) identified by a monoclonal antibody generated against chick embryo neural retina. Biochem. Biophys. Res. Commun. 1999, 257, 480487. (17) Albert, K. A.; Walaas, S. I.; Wang, J. K. T.; Greengard, P. Widespread occurrence of “87 kDa”, a major specific substrate for protein kinase C. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 2822-2826. (18) Zolessi, F. R.; Arruti, C. Sustained phosphorylation of MARCKS in differentiating neurogenic regions during chick embryo development. Brain Res. Dev. Brain Res. 2001, 130, 257-267. (19) Stumpo, D. J.; Bock, C. B.; Tuttle, J. S.; Blackshear, P. J. MARCKS deficiency in mice leads to abnormal brain development and perinatal death. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 944-948. (20) Aigner, L.; Caroni, P. Depletion of 43-kD growth-associated protein in primary sensory neurons leads to diminished formation and spreading of growth cones. J. Cell Biol. 1993, 123, 417429. (21) Peter, J. F.; Tomer, K. B. A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking. Anal. Chem. 2001, 73, 4012-4019. (22) Graff, J. M.; Stumpo, D. J.; Blackshear, P. J. Molecular cloning, sequence, and expression of a cDNA encoding the chicken myristoylated alanine-rich C kinase substrate (MARCKS). Mol. Endocrinol. 1989, 3, 1903-1906. (23) Songyang, Z.; Lu, K. P.; Kwon, Y. T.; Tsai, L-H.; Filhol, O.; Cochet, C.; Brickey, D. A.; Soderling, T. R.; Bartleson, C.; Graves, D. J.; Demaggio, A. J.; Hoekstra, M. F.; Blenis, J.; Hunter, T.; Cantley, L. C. A structural basis for substrate specificities of protein Ser/ Thr kinases: primary sequence preference of Casein Kinases I and II, NIMA, Phosphorylase Kinase, Calmodulin-Dependent Kinase II, CDK5, and Erk1. Mol. Cell Biol. 1996, 16, 6486-6493. (24) Holmes, J. K.; Solomon, M. J. A predictive scale for evaluating cyclin-dependent kinase substrates. A comparison of p34cdc2 and p33cdk2. J. Biol. Chem. 1996, 271, 25 240-25 246. (25) Scho¨nwasser, D. C.; Palmer, R. H.; Herget, T.; Parker, P. J. p 42 MAPK phosphorylates 80 kDa MARCKS at Ser-113. FEBS Lett. 1996, 395, 1-5. (26) Zhao, Y.; Muir, T. W.; Kent, S. B. H.; Tischer, E.; Scardina, J. M.; Chait, B. T. Mapping protein-protein interactions by affinitydirected mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4020-4024. (27) Yu, L.; Gaskell, S. J.; Brookman, J. L. Epitope mapping of monoclonal antibodies by mass spectrometry: identification of protein antigens in complex biological systems. J. Am. Soc. Mass Spectrom. 1998, 9, 208-215.

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