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What does the term 'modified residue position' in phosphorylation mean?

What does the term 'modified residue position' in phosphorylation mean?


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Does it mean the position of the amino acid in the protein sequence, or something else?

For example, I came across the phrase "S 368 phosphoryation" where S is the modified residue and 368 is the modified residue position. What does it all mean?


You are correct, the368stands for the position of the amino acid in the protein's sequence - this particular serine is the 368th residue in the protein counting from the amino-terminal end.


Post-translational modification

Post-translational modification (PTM) refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signaling, as for example when prohormones are converted to hormones.

Post-translational modifications can occur on the amino acid side chains or at the protein's C- or N- termini. [1] They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphate. Phosphorylation is a very common mechanism for regulating the activity of enzymes and is the most common post-translational modification. [2] Many eukaryotic and prokaryotic proteins also have carbohydrate molecules attached to them in a process called glycosylation, which can promote protein folding and improve stability as well as serving regulatory functions. Attachment of lipid molecules, known as lipidation, often targets a protein or part of a protein attached to the cell membrane.

Other forms of post-translational modification consist of cleaving peptide bonds, as in processing a propeptide to a mature form or removing the initiator methionine residue. The formation of disulfide bonds from cysteine residues may also be referred to as a post-translational modification. [3] For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain the resulting protein consists of two polypeptide chains connected by disulfide bonds.

Some types of post-translational modification are consequences of oxidative stress. Carbonylation is one example that targets the modified protein for degradation and can result in the formation of protein aggregates. [4] [5] Specific amino acid modifications can be used as biomarkers indicating oxidative damage. [6]

Sites that often undergo post-translational modification are those that have a functional group that can serve as a nucleophile in the reaction: the hydroxyl groups of serine, threonine, and tyrosine the amine forms of lysine, arginine, and histidine the thiolate anion of cysteine the carboxylates of aspartate and glutamate and the N- and C-termini. In addition, although the amide of asparagine is a weak nucleophile, it can serve as an attachment point for glycans. Rarer modifications can occur at oxidized methionines and at some methylenes in side chains. [7]

Post-translational modification of proteins can be experimentally detected by a variety of techniques, including mass spectrometry, Eastern blotting, and Western blotting. Additional methods are provided in the external links sections.


Phosphorylation of Calcineurin B-like (CBL) Calcium Sensor Proteins by Their CBL-interacting Protein Kinases (CIPKs) Is Required for Full Activity of CBL-CIPK Complexes toward Their Target Proteins*

Calcineurin B-like proteins (CBLs) represent a family of calcium sensor proteins that interact with a group of serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). CBL-CIPK complexes are crucially involved in relaying plant responses to many environmental signals and in regulating ion fluxes. However, the biochemical characterization of CBL-CIPK complexes has so far been hampered by low activities of recombinant CIPKs. Here, we report on an efficient wheat germ extract-based in vitro transcription/translation protocol that yields active full-length wild-type CIPK proteins. We identified a conserved serine residue within the C terminus of CBLs as being phosphorylated by their interacting CIPKs. Remarkably, our studies revealed that CIPK-dependent CBL phosphorylation is strictly dependent on CBL-CIPK interaction via the CIPK NAF domain. The phosphorylation status of CBLs does not appear to influence the stability, localization, or CIPK interaction of these calcium sensor proteins in general. However, proper phosphorylation of CBL1 is absolutely required for the in vivo activation of the AKT1 K + channel by CBL1-CIPK23 and CBL9-CIPK23 complexes in oocytes. Moreover, we show that by combining CBL1, CIPK23, and AKT1, we can faithfully reconstitute CBL-dependent enhancement of phosphorylation of target proteins by CIPKs in vitro. In addition, we report that phosphorylation of CBL1 by CIPK23 is also required for the CBL1-dependent enhancement of CIPK23 activity toward its substrate. Together, these data identify a novel general regulatory mechanism of CBL-CIPK complexes in that CBL phosphorylation at their flexible C terminus likely provokes conformational changes that enhance specificity and activity of CBL-CIPK complexes toward their target proteins.

This work was supported by Deutsche Forschungsgemeinschaft grants within the frame of the Research Unit 964 (to D. B., M. H., and J. K.).

Present address: Division of Biological Sciences, Cell and Developmental Biology Section and Center of Molecular Genetics, University of California San Diego, 9500 Gilman Dr., 0116, La Jolla, CA 92093-0116.

M. Rehers, Hashimoto, and J. Kudla, unpublished results.


Author Summary

Parkinson’s disease is characterized by loss of dopaminergic neurons in midbrain and the presence of αSyn protein inclusions. Human αSyn mimics the disease pathology in yeast resulting in cytotoxicity and aggregate formation. αSyn is abundantly phosphorylated at serine S129 and possesses four tyrosines (Y39, Y125, Y133, and Y136) that can be posttranslationally modified by nitration or phosphorylation. The consequence of each of these possible modifications is still unclear. Nitration as consequence of oxidative stress is a hallmark for neurodegenerative diseases. Here, we addressed the molecular mechanism, how tyrosine posttranslational modifications affect αSyn cytotoxicity. Tyrosine nitration can contribute to αSyn toxicity or can be part of a cellular salvage pathway when di-tyrosine-crosslinked dimers are formed. The Y133 residue, which can be either phosphorylated or nitrated, determines whether S129 is protectively phosphorylated and αSyn inclusions are cleared. This interplay with S129 phosphorylation demonstrates a dual role for C-terminal tyrosine residues. Yeast flavohemoglobin Yhb1 and its human counterpart neuroglobin NGB protect cells against cytotoxicity and aggregate formation. These novel insights into the molecular pathways responsible for αSyn cytotoxicity indicate NGB as a potential target for therapeutic intervention in PD.

Citation: Kleinknecht A, Popova B, Lázaro DF, Pinho R, Valerius O, Outeiro TF, et al. (2016) C-Terminal Tyrosine Residue Modifications Modulate the Protective Phosphorylation of Serine 129 of α-Synuclein in a Yeast Model of Parkinson's Disease. PLoS Genet 12(6): e1006098. https://doi.org/10.1371/journal.pgen.1006098

Editor: Bingwei Lu, Stanford University School of Medicine, UNITED STATES

Received: December 22, 2015 Accepted: May 10, 2016 Published: June 24, 2016

Copyright: © 2016 Kleinknecht et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper.

Funding: This work was funded by the DFG Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB). RP is supported by a PhD fellowship from FCT Portugal (SFRH/BD/80884/2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Discussion

Binding between intrinsically disordered and ordered proteins is an important field and molecular dynamics simulations is one of the most suitable methods to study it, especially to answer mechanistic questions. We have described conformational changes of an intrinsically disordered protein, AANAT, before and after its phosphorylation and during the binding to 14-3-3ζ, an ordered scaffold protein. For statistical robustness, we run three independent experiments giving equivalent results and conclusions (each one including three 110 ns simulations with and without Thr31 phosphorylation, summing up to total simulation time of more than 300 ns × 2). Full details and plots of all quantities measured in each simulation run are included in the section Supplementary Information. In a previous work, we have theoretically proposed the presence of a metastable pseudo-native complex between these two proteins, and described the role of anchor residues in the process. Glu87 contributes to the binding process between AANAT and 14-3-3ζ via specific intermolecular contacts, and a loss of affinity is observed when mutating this specific residue to Ala (Fig. 1). However, our stopped-flow experiment showed no indications of less affinity complexes during binding of Glu87Ala mutant AANAT and 14-3-3ζ, therefore the dynamics of this process towards the final stable complex remained unexplained.

Some articles were published describing the binding mechanisms using the model system KID domain of the transcription factor CREB by free molecular dynamics. However, for the time being, equilibrium all-atom molecular dynamics simulations of coupled folding and binding are out of reach for the majority of researchers. It is clear then that the best opportunity for obtaining atomic-resolution information on coupled folding and binding relies on the use of enhanced sampling methods 15 , like harmonically restrained potentials. KID phosphorylation induces a minor shift in the equilibrium distribution of folded regions. Binding of kinetically locked and constrained regions can give other transient interactions time to form, potentially rising the number of productive binding events and thus increasing the affinity for the binding partner. Similarly, kinetically locked regions that participate in molecular recognition could frustrate the unbinding process. This would result in a shift of the binding equilibrium towards the bound state 16,17 . Another example is the phosphorylation of KH1 domain of KSR, which unfolds and creates a site for 14-3-3ζ binding 18 . KH1 domain interacts with 14-3-3ζ only when it is phosphorylated and unfolded. AKT phosphorylation of KH1 is responsible for its unfolding, which forms the binding site for 14-3-3ζ. Structural rearrangement upon phosphorylation is a common regulatory mechanism 19 , but there are no reported examples, to the best of our knowledge, of a complete protein-protein binding analysis.

Our results indicate that phosphorylation on 14-3-3ζ target proteins has broad implications during the binding process and not only in the stabilization of final complexes. AANAT has two canonical sites, one in the N-terminal and the other in the C-terminal of the protein. However, the complex crystal structure can only be solved when the second C-terminal binding site (considered of lower affinity) has been removed 46 . Here, we used the elementary unit (one monomer of 14-3-3ζ and C-terminal truncated AANAT) to mechanistically analyze how phosphorylation affects binding between AANAT and 14-3-3ζ. In order to reduce computational costs, we applied a protocol divided in three regions with a total of 110 ns of simulation time. For statistical robustness, we run three independent simulations giving identical results and conclusions. In the first and last 10 ns, we applied a harmonic potential to induce the formation of the pseudo-native and native complexes, respectively. In region II, we run 90 ns of unrestrained dynamics. We clearly observed that phosphorylation of Thr31 gives this residue (and the surrounding amino acids) the ability to explore more conformations following a mechanism called ‘fly-casting’ 5 . During this process, the capture radius is increased and the binding process enhanced. We calculated the capture radius as the radius of gyration of the residues of interest (see equation 2) and observed a 40% increase for Thr31 (see Fig. 7b). Surprisingly, this phosphorylation has also implications in 14-3-3ζ, mainly through long-range interactions. We observed that the major regulatory region on 14-3-3 (α-helix 9) is also affected by Thr31 phosphorylation in AANAT as the movement of this region (α-helix 9) is also increased (Fig. 7a). These enhanced movements allow the protein-protein system to reach a more compact final complex at the end of our simulations. To measure this, 3D-SASA maps are used here as semi-quantitative estimates of the desolvation free energy upon protein complexation 20 . For our two conditions (AANAT and AANAT*) the final complex is

3.5 Å with respect to the complex crystal structure, however, in the AANAT situation the final structure has much more solvent exposed surface than in the AANAT* one, suggesting a less stable conformation (see Fig. 7c,d).

The initial association between proteins containing IDR is often strongly dependent on ionic strength, demonstrating that specific or non-specific charge-charge interactions govern the rate of association 21 . The electrostatic field guides the protein to its target and thus accelerates the binding rate. Protein flexibility also facilitates binding via the fly-casting mechanism 22 . Our results strongly agree with this interpretation. 14-3-3ζ is a protein with a net negative charge 23 and a positive hot spot where the phosphoryl group of its partners docks, demonstrating that phosphorylation of Thr31 on AANAT is the key through the binding process.

The current vision of protein phosphorylation has a question with no evident answer: is single residue phosphorylation mediated by a scaffold protein? and in particular, are 14-3-3 proteins only readers of the modification? As it has been shown in this work, phosphorylation has effects on both the partner and the reader protein in a wide sense. We propose that phosphorylation must occur before protein-protein binding and that 14-3-3 proteins only read the modification and do not participate in it.


Introduction

At least two distinct populations of secretory vesicles mediate the regulated exocytotic release of neurotransmitters. Synaptic vesicles (SVs) appear almost exclusively in nerve terminals where they cluster at the synaptic cleft (Calakos and Scheller 1996). In contrast, large dense core vesicles (LDCVs) appear throughout the cell, and release transmitter more slowly and in response to different stimuli than SVs (De Camilli and Jahn 1990 Martin 1994). Although SVs are generally considered to contain classical transmitters, whereas LDCVs store neural peptides, both types of regulated secretory vesicles contain monoamines, indicating the potential for release of a single transmitter through two distinct mechanisms (Thureson-Klein 1983 Bruns and Jahn 1995). Since classical transmitters, including monoamines, are synthesized in the cytoplasm, the subcellular location of the transporters required for packaging them into secretory vesicles determines the site of vesicular storage, and hence, the mode of exocytotic release.

Secretory vesicles exhibit four distinct neurotransmitter transport activities, including one for monoamines, a second for acetylcholine (ACh), a third for γ-aminobutyric acid (GABA), and a fourth for glutamate (Liu and Edwards 1997b). All of these activities depend on the H + electrochemical gradient across the vesicle membrane, and exchange lumenal protons for cytoplasmic transmitter (Schuldiner et al. 1995). Pharmacologic manipulation of vesicular transport indicates the importance of these activities for behavior. The antihypertensive drug reserpine inhibits vesicular monoamine transport and induces a syndrome resembling depression (Frize 1954). In addition, amphetamines induce efflux from vesicular monoamine stores (Sulzer et al. 1995) and can cause psychosis. Thus, changes in vesicular transport activity have the potential to influence behavior, but the extent to which they undergo regulation has remained unknown.

Molecular cloning has begun to identify the proteins responsible for neurotransmitter transport into secretory vesicles. One family of proteins includes two vesicular monoamine transporters (VMATs) and the vesicular acetylcholine transporter (VAChT). VMAT1 is expressed principally in peripheral, nonneural tissues and VMAT2 is expressed by central monoamine neurons (Varoqui and Erickson 1997 Liu and Edwards 1997b). Consistent with the observed release of monoamines from both SVs and LDCVs, the VMATs occur on multiple populations of secretory vesicles. In brain, VMAT2 localizes preferentially to LDCVs, but also resides on SVs, as well as tubulovesicular structures in the cell body and dendrites of central dopamine neurons (Nirenberg et al. 1995, Nirenberg et al. 1996). Similarly, VMAT2 resides predominantly on LDCVs rather than the synaptic-like microvesicles (SLMVs) present in PC12 cells (Liu et al. 1994 Erickson et al. 1996). In contrast, VAChT localizes predominantly to SVs in brain (Gilmor et al. 1996 Weihe et al. 1996) and to lighter membranes, including the SLMVs in PC12 cells (Liu and Edwards 1997a Varoqui and Erickson 1998). However, VAChT also occurs at lower levels on LDCVs in PC12 cells (Liu and Edwards 1997a) and in neurons (Lundberg et al. 1981 Agoston and Whittaker 1989). Thus, two closely related vesicular neurotransmitter transporters localize to distinct populations of secretory vesicles that differ in their mode of release.

To understand how VMAT2 and VAChT localize to different secretory vesicles, we have sought to identify their sorting signals. As integral membrane proteins, the transporters presumably depend on signals in their cytoplasmic domains that interact with a cytosolic sorting machinery. We have found previously that dileucine motifs located COOH-terminal to transmembrane domain (TMD) 12 of both VMAT2 and VAChT (see Fig. 1 A) mediate internalization of the transporters from the plasma membrane (Tan et al. 1998). Dileucine motifs implicated in the endocytosis of certain other proteins require the presence of acidic residues at positions −4 and −5 relative to the two leucines (Pond et al. 1995 Dietrich et al. 1997) and the VMATs contain highly conserved glutamates at both of these positions. However, replacement of these glutamates with alanine does not impair the internalization of VMAT2 (Tan et al. 1998). Since dileucine motifs mediate trafficking at multiple sites in the secretory pathway, these residues may influence other events, distinct from endocytosis. Although VAChT, like the VMATs, contains a glutamate at −4 relative to the dileucine, it contains a serine at −5, raising the possibility that the charge of this residue accounts for the localization of the transporters to distinct populations of secretory vesicles. In addition, the presence of a serine at −5 relative to the dileucine in VAChT suggests that phosphorylation of this residue may influence the sorting of the transporter. Indeed, phosphorylation of serine residues at −5 relative to the dileucine motifs in CD4 (Shin et al. 1991) and CD3γ (Dietrich et al. 1994) dramatically influences the trafficking of these proteins.

We now report that the serine at −5 relative to the dileucine motif in VAChT (Ser-480) undergoes phosphorylation by a calcium-dependent isoform of protein kinase C (PKC). Replacement of Ser-480 by alanine, which prevents phosphorylation, reduces the expression of VAChT on LDCVs relative to the wild-type protein. In contrast, substitution of Ser-480 by glutamate, to mimic the sequence of VMAT2 at this position and the phosphorylation event, increases the expression of VAChT on LDCVs. Consistent with the importance of acidic residues upstream of a dileucine motif in sorting to LDCVs, replacement of Glu-478 and -479 upstream of the dileucine-like motif in VMAT2 reduces localization to LDCVs. The results provide some of the first information about signals involved in sorting to different classes of secretory vesicles. In addition, they suggest that phosphorylation regulates the targeting of VAChT to LDCVs, providing a potential mechanism to regulate transmitter release.


What does the term 'modified residue position' in phosphorylation mean? - Biology

The p21-activated kinases (Paks) serve as effectors of the Rho family GTPases Rac and Cdc42. The six human Paks are divided into two groups based on sequence similarity. Group I Paks (Pak1 to -3) phosphorylate a number of substrates linking this group to regulation of the cytoskeleton and both proliferative and anti-apoptotic signaling. Group II Paks (Pak4 to -6) are thought to play distinct functional roles, yet their few known substrates are also targeted by Group I Paks. To determine if the two groups recognize distinct target sequences, we used a degenerate peptide library method to comprehensively characterize the consensus phosphorylation motifs of Group I and II Paks. We find that Pak1 and Pak2 exhibit virtually identical substrate specificity that is distinct from that of Pak4. Based on structural comparisons and mutagenesis, we identified two key amino acid residues that mediate the distinct specificities of Group I and II Paks and suggest a structural basis for these differences. These results implicate, for the first time, residues from the small lobe of a kinase in substrate selectivity. Finally, we utilized the Pak1 consensus motif to predict a novel Pak1 phosphorylation site in Pix (Pak-interactive exchange factor) and demonstrate that Pak1 phosphorylates this site both in vitro and in cultured cells. Collectively, these results elucidate the specificity of Pak kinases and illustrate a general method for the identification of novel sites phosphorylated by Paks.

This research was supported in part by an American Association for Cancer Research-Fox Chase Cancer Center Career Development Award in Translational Cancer Research, by Department of Defense Neurofibromatosis Research Program Grant W81XWH-05-1-0200, and by a grant from the Pennsylvania Department of Health (all to J. R. P.). Additional support was provided by the National Institutes of Health Grant CA006927 and by an appropriation from the Commonwealth of Pennsylvania to Fox Chase Cancer Center. The Structural Genomics Consortium is a registered charity (number 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, the Canadian Foundation for Innovation, VINNOVA, the Knut and Alice Wallenberg Foundation, the Swedish Foundation for Strategic Research, and Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2 and Fig. 1.

Supported by funding from NCI, National Institutes of Health (NIH), Grant T32 CA009035.

Present address: Division of Biology, Kansas State University, Manhattan, KS 66502.


Supporting Information

This provides a detailed description of secondary structural and RMSF analysis for KLHL3 (Supporting Information Fig. S1), electrostatic potential of KLHL3 for the equilibrated simulations (Supporting Information Fig. S2), time-dependent hydrogen bonds formed between the key residues of KLHL3 and the acidic motif of WNK4 (Supporting Information Fig. S3), comparison of the interaction energy for residues of KLHL3 involved in hydrophobic interactions between Kelch domain and acidic motif of WNK4 (Supporting Information Fig. S4), and distance between the acidic motif and the Kelch domain (Supporting Information Fig. S5) as well as a comparison between the distances in the presence of pS433 and protonated pS433 (Supporting Information Fig. S6).


Author contributions

DB, FDR, and AB designed the experiments of this study. DB, ES, AA, AT, CB, and AN conducted the experiments. DB, FDR, and AB performed data analysis and critical discussion of the results. DB, DR, SP, FDR, and AB contributed to the writing and editing of the manuscript. All authors approved the final draft of the manuscript.

Filename Description
mol212881-sup-0001-FigS1.jpgJPEG image, 1.3 MB Fig. S1. Transfection efficiency evaluation. HEK-293 cells transfected for 48 h with empy-vector (CTRL), wt-p27 and G9R-p27 were stained with anti-p27 mAb and fluorescence-tagged secondary antibodies and analyzed by flow cytometry using a FACScalibur. Calculations were done over 30 000 events. M2 includes wt- and mutated p27 expressing cells, corresponding at least to 50% of the whole cell populations. M1 comprises cells with a very low level of fluorescence corresponding to endogenous p27 staining.
mol212881-sup-0002-FigS2.jpgJPEG image, 1.8 MB Fig. S2. Spheroid formation ability of cells expressing wt-p27 and G9R-p27. LN-229 glioblastoma cells transfected the day before with empty vector (CTRL) or plasmids encoding WT-, and G9R-p27 were seeded in matrigel for 3D spheroid-based tumor invasion assay. Details are reported under ‘Materials and methods’. Cultures were observed under light microscope and images were taken at 4, 5 and 6 days after seeding. The experiment was repeated three times, while the figure reports the results of two replicates for each time point. On the right, images obtained after 6 days inclusion at higher magnification: G9R-expressing cultures show the presence of cells that appear detaching from spheres (protruding cells).
mol212881-sup-0003-FigS3.jpgJPEG image, 1.5 MB Fig. S3. Spheroid formation ability of PC-3 cells expressing wt-p27 and G9R-p27. PC-3 cells transfected the day before with empty vector (CTRL) or plasmids encoding WT-, and G9R-p27 were seeded in matrigel for 3D spheroid-based tumor invasion assay. Details are reported under ‘Materials and methods’. Cultures were observed under light microscope and images were taken at 6 days after seeding. The experiment was repeated three times, while the figure reports the results of two replicates. On the right and on the left, images were obtained at different magnification. On the center, the diameter of the colonies obtained was measured using the scale bar (50 µm) as reference. The results shown are the mean of 3 determinations obtained on three independent experiments and standard deviation is showed. Data were analyzed by Student's t test. *P < 0.05.
mol212881-sup-0004-FigS4.jpgJPEG image, 961.2 KB Fig. S4. Apoptosis analysis of cells expressing G9R-p27 compared to wt-p27. PC-3 cells were transfected for 48 h with pcDNA3.0 empty vector, and pcDNA3.0 encoding p27, or G9R-p27. Then, cells were treated for 18 h with 1 µ m staurosporine. Cells were collected and processed with Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit according to manufacturer's indications. The control of this experiment is made by cells transfected with empty vector and treated with staurosporine (STAUROSPORINE) as reported under ‘Materials and methods’. Cell apoptosis was detected by flow cytometry using a FACScalibur and calculated analyzing 50 000 events. Upper right (UR) quadrant includes apoptotic cells.
mol212881-sup-0005-FigS5.jpgJPEG image, 839.8 KB Fig. S5. Bidimensional analysis of transfected mutants of G9R-p27. (A) 2D/WB analysis of cell extracts of PC-3 cells transfected with pcDNA3.0 plasmids encoding S10A/G9R-p27, S10A/T187A/G9R-p27 [*T187A], S10A/T198V/G9R-p27 [*T198V], S10A/T157A/G9R-p27 [*T157A], and S10A/Y(74,88,89)F/G9R-p27 [*Y(74,88,89)F] on the left, and plasmids encoding S10A/G9R-p27, S7A/S10A/G9R-p27 [*S7A], S183A/S10A/G9R-p27 [*S183A], S83A/S10A/G9R-p27 [*S83A] on the right. After blotting, the filters were analyzed by mAb anti-p27. Signals 0 and 1correspond to unmodified and 1Pi-protein, respectively. (B) HeLa cells were transfected with pcDNA3.0 plasmids encoding G9R-p27, S10A/G9R-p27 [*S10A], and S12A/G9R-p27 [*S12A]. Cell extracts were prepared and analyzed by 2D/WB. After blotting, the filters were analyzed by mAb anti-p27. Signals 0, 1, and 2 correspond to unmodified, 1Pi- and 2Pi-protein, respectively. (C) On the left. 2D/WB analysis of cell extracts of K562 cells transfected with pcDNA3.0 plasmids encoding G9R-p27 and S12A/G9R-p27 [*S12A]. After blotting, the filters were analyzed by mAb anti-p27. Signals 0, 1, and 2 correspond to unmodified, 1Pi- and 2Pi-protein, respectively. On the right. 2D/WB analysis of cell extracts of SH-SY5Y cells transfected with pcDNA3.0 plasmids encoding G9R-p27. After blotting, the filters were analyzed by mAb anti-p27. Signals 0, 1, and 2 correspond to unmodified, 1Pi- and 2Pi-protein, respectively. (D) On the left. 2D/WB analysis of cell extracts of K562 cells transfected with pcDNA3.0 plasmids encoding p27 protein, and its derivatives S12A/p27 [*S12A], and S12D/p27 [*S12D]. On the right. The histograms report the intensity percentage of each signal (unmodified, 1Pi-isoforms) relative to the total for p27 and its derivative mutant proteins. The intensity of the specific signals was evaluated using TotalLab CLIQS gel image analysis Software. The data shown are the results of three independent experiments. Bars represent standard deviation. Data were analyzed by Student's t test. **P < 0.01.
mol212881-sup-0006-FigS6.jpgJPEG image, 1.3 MB Fig. S6. Effect of CDK2 on the nuclear and cytosolic localization of G9R-p27. (A) Different population of MEF cells were investigated for confirming the absence of CDK2 and CDK4 protein. Immortalized wild type MEFs, and MEFs lacking CDK4, or CDK2 or both CDK4 and CDK2 were cultured as in Materials and methods. Then, the nuclear and cytosol compartments were prepared and analyzed for CDK2 and CDK4 by WB and specific antibodies. The filters were also analyzed for HDAC and PKM2 content by specific antibodies in order to confirm equal loading and compartment separation. (B) Upper figure. pcDNA3.0 plasmid encoding G9R-p27 was transfected in different MEF populations, namely MEF immortalized cells, CDK4 −/− MEFs, CDK2 −/− and CDK4 −/− CDK2 −/− cells. After 24 h, nuclear and cellular compartments were prepared and analyzed by WB employing mAb anti-p27. HDAC1 was investigated for evaluating loading amount and nuclear purity. Lower figure. Three experiments similar to that reported on the top were performed. The percentage of nuclear and cytosolic protein was evaluated by imagej software. On the basis of determined data, the showed histograms were constructed. Error bars represent the standard error of the mean of the experiments. (C) pcDNA3.0 plasmids encoding p27 and G9R-p27 were transfected in parental and CDK2 −/ CDK4 − MEFs. After 24 h, nuclear and cytosol extracts were prepared and analyzed by 2D/WB with mAb anti-p27. For the nuclear extracts, images at different film exposition times are reported. (D) S10/S12/T187A/G9R-p27 protein was prepared from PC-3 transfected cells. The partially purified protein was incubated with recombinant CDK2 for 40 min. The assay mixtures at time 0 and after 40 min were analyzed by 2D/WB.

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Materials and Methods

Cell cultures

Vero monkey kidney, epithelial HEK293T or LLCPK1 cells were grown in Dulbecco's modified Eagle's medium. Jurkat T lymphoid cells were maintained in RPMI 1640 medium (Bio Whittaker Europe, Verviers, Belgium). Media were supplemented with 10% fetal calf serum and cells were grown at 37°C in 5% CO2.

Antibodies and reagents

Polyclonal rabbit IgGs against L-plastin have been previously characterised (Lapillonne et al., 2000). Mouse monoclonal anti-vinculin antibody was a kind gift of M. Glukhova (Institut Curie, Paris, France) anti-cortactin antibody was purchased from Upstate (TE Huissen, The Netherlands) and anti-vimentin antibody from Santa Cruz Biotechnology (Tebu-Bio, Boechout, Belgium). Polyclonal anti-Ser5-P antibody against L-plastin phosphorylated at Ser5 was raised against a peptide encoding L-plastin residues 2-17 in which Ser5 was phosphorylated (ARGS(P)VSDEEMMELREA). Rabbit antiserum was purified by negative affinity on non-phosphorylated peptide followed by positive affinity on the phosphorylated peptide. The monoclonal VII-E-7 antibody against the 13 C-terminal residues of the Sendai virus L protein (sv-tag) was a kind gift of J. Neubert (Max-Planck-Institut für Biochemie, Martinsried, Germany). The rabbit polyclonal antibody against the same sequence was described previously (Arpin et al., 1994). The anti-rabbit IgG antibody coupled to horseradish peroxidase was purchased from Amersham Biosciences (Roosendaal, The Netherlands). Cy2-conjugated goat anti-rabbit IgG was purchased from Jackson Immuno-Research Laboratories (De Pinte, Belgium). Alexa Fluor 350- or 594-coupled phalloidin and secondary antibodies were purchased from Molecular Probes (Invitrogen, Merelbeke, Belgium). β-G-actin was purchased from Cytoskeleton (Boechout, Belgium). PKA catalytic subunit, PKA activator 8-Bromo-cAMP, PKA inhibitors H89 and KT5720 and PKC inhibitor GF109 and Gö6796 were purchased from Calbiochem (Leuven, Belgium). Forskolin was obtained from Sigma (Bornem, Belgium). Lipofectin reagent was purchased from Invitrogen.

Site-directed mutagenesis of cDNAs

To mutate Ser5 into Ala or a Glu by a PCR-based approach, Mut5A (5′-AAAAATGGCCAGAGGA GCA GTGTCC-3′), Mut5E (5′-AAAAATGGCCAGAGGA GAA GTGTCC-3′) sense primers corresponding to L-plastin nucleotides 4-21 were used. Underlined sequences indicate mutated amino acids. The common reverse primer (3′-CTTCCCCTCCTTCAGGTCCTCAGC-5′) was complementary to bases 802-780 of L-plastin cDNA and flanked at its 5′-end by an NcoI site. The PCR fragment containing the mutation was used to replace the corresponding fragment of the L-plastin cDNA inserted in the pCB6 expression vector, downstream of the cytomegalovirus promoter. The Ser5Glu and Ser5Ala cDNAs were also cloned into the BamHI-EcoRI sites of the pGEX-2T expression vector. Mutated DNAs were verified by sequencing.

Recombinant proteins

Wild-type human L-plastin, as well as the Ser5Glu and Ser5Ala variants of L-plastin were produced in E. coli from the pGEX-2T expression vector and purified as described previously (Arpin et al., 1994). The concentration of thrombin-cleaved proteins was determined according to the Bradford method (BioRad, Nazareth, Belgium) and by SDS polyacrylamide gel electrophoresis (PAGE) using a BSA protein standard curve.

Transient expression of cDNAs in cells

Five or 10 μg of cDNA encoding wild-type human L-plastin or L-plastin variants was transfected respectively into HEK239T cells using calcium phosphate procedure (Chen and Okayama, 1988) or into 5×10 6 Vero cells by electroporation at 240 volt and 950 μF (Toneguzzo et al., 1986). LLCPK1 cells were transfected by Lipofectin.

Treatment of cells with pharmacological agents

Cells were washed with PBS and resuspended in HBSS ++ (1× Hanks' buffered salt solution) containing 20 mM Hepes, 0.5 mM Mg 2+ and 1.0 mM Ca 2+ . Jurkat T lymphoid cells or HEK293T cells were treated with PKA or PKC activators or inhibitors as indicated in the figure legends and described (Wang and Brown, 1999). After treatment, cells were lysed and processed for immunoblotting analysis.

Analysis of L-plastin or L-plastin variants by immunoblotting

Cells were lysed for 30 minutes in ice-cold RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% NP40 and 1% Na-deoxycholate) containing a cocktail of protease and phosphatase inhibitors. Lysates were cleared by centrifugation in a microfuge at 16,000 g for 10 minutes at 4°C, and the protein concentration was determined using a modified Lowry method or Bradford assay (BioRad).

Total cell lysates (20 μg of protein) were separated by PAGE under reducing conditions and transferred onto nitrocellulose membrane (Schleicher & Schuell) using a semi-dry transblot apparatus. Primary antibodies indicated in figure legends were revealed by using secondary antibodies coupled to horseradish peroxidase and enhanced chemiluminescence (ECL). In some experiments, the membrane was stripped as previously described (Janji et al., 1999).

Indirect immunofluorescence

Transfected cells were fixed with 3% paraformaldehyde and processed for immunofluorecence labelling as described previously (Friederich et al., 1999). Labelled cells were analysed by epifluorescence microscopy (Leica DMRX microscope, HCX PL APO ×63 or ×100) or a Zeiss laser scanning confocal microscope (LSM-510 Meta). Images were acquired with a linear CCD camera (micromax, Princeton Instruments) and analysed with Metaview or Metamorph software (Universal Imaging Cooperation)

Detergent extraction of cells

Transfected Vero cells were plated onto glass coverslips. After 48 hours, coverslips were cut in halves. One half was directly processed for indirect immunofluorescence, while the other was detergent-extracted with 0.5% Triton X-100 for 16 seconds at 20°C, as previously described (Arpin et al., 1994 Friederich et al., 1992) and then processed for immunofluorescence labelling. For quantification, number of cells with detectable sv-tag labelling per 100 cells were determined for each half coverslip, yielding quantitative information on the transfection efficiency and on the resistance of transfected proteins to detergent extraction.

Phosphorylation of L-plastin in vitro

L-plastin protein was phosphorylated using PKA catalytic subunit (specific activity ⩾750 units/μg of protein) at 30°C in a reaction mixture containing 20 mM Tris-HCl pH 7.4, 20 mM MgCl2, 10 mM dithiothreitol and 100 μM ATP. To monitor phosphorylation, aliquots were removed, the reaction was stopped by addition of boiling SDS-sample buffer and proteins were analysed by immunoblotting following the ECL detection method (Amersham Bioscience). To analyse F-actin binding capacity of L-plastin, aliquots from the reaction were added to G-actin and processed as described below.

Actin binding and bundling assays

G-actin was polymerised overnight at 4°C or for 4 hours at room temperature in the presence of L-plastin variants in polymerisation buffer (100 mM KCl, 1 mM MgCl2, 1 mM ATP, 0.5 mM EGTA, 50 mM sodium phosphate buffer, pH 7.0) as indicated in figure legends. Sedimentation of actin filaments and L-plastin was achieved by high-speed centrifugation at 200,000 g for 30 minutes. To sediment actin bundles, samples were centrifuged for 15 minutes at 12,000 g (Glenney, Jr et al., 1981). Proteins in pellets and supernatants were separated by SDS-PAGE. Coomassie-stained protein bands were scanned and densities were quantified using BioCapt and Bio-PROFIL Bio 1D software (Windows applications) or LabImage software 2.7.2 (Kapelan Bio-Imaging Solution).

Transmission electron microscopy

Samples prepared for the above-described bundling assay were analysed by transmission electron microscopy. Aliquots were removed from the mixture before centrifugation and immediately negatively stained with 1% uranyl acetate. Electron micrographs were obtained using a Philips CM 120 microscope at a magnification of 28,000× or 45,000×.

Collagen invasion assays

Gels were prepared in a six-well plate from a collagen-type-I solution (Upstate Biotechnology, Lake Placid, NY). Cells (1×10 5 ) were incubated on top of the gels for 24 hours at 37°C. HEK293T cells inside the gel were scored with a phase contrast microscope controlled by a computer program (Bracke et al., 2001 De Corte et al., 2002). Invasive and superficial cells were counted in 12 fields of 0.157 mm 2 . The invasion index is the percentage of cells invading the gel over the total number of cells counted. DHD-FIB rat colon myofibroblasts were used as a positive control. Experiments were performed in triplicate. Mean values and standard deviations were calculated.

Fast aggregation assay

Cell-cell adhesion was numerically evaluated in an aggregation assay as described earlier (Boterberg et al., 2001). Briefly, single-cell suspensions of HEK293T cells were prepared according to an N-cadherin-saving procedure and allowed to aggregate on a Gyrotory shaker at 80 rpm for 30 minutes in aggregation buffer (1.25 mM Ca 2+ , 0.1 mg DNase /ml, 10 mM Hepes and 0.1% BSA) and equilibrated at physiological pH and osmolarity. Cell aggregation was measured with an LS particle size analyzer (LS 200, Coulter Electronics) after 0 and 30 minutes of aggregation. The relative volume in function of the particle size was used as an index of aggregation. Semi-quantitative evaluation of Fast Aggregation (FA) was determined as follow. Less than 20 μm, no aggregates I between 20 and 100 μm, no aggregates II between 100 and 1000 μm, aggregates III more than 1000 μm, aggregates IV.

Wound healing assay

HEK293T cells (8×10 5 ) were seeded into six-well cell culture plates and 18 hours after seeding, cells were transfected with cDNA constructs. After 48 hours, a wound was made by scratching a line in a confluent monolayer. Cell debris was removed by washing the cells with serum-free medium. Migration of cells into the wound was then observed at different time points. Cells were followed for 24 hours.