Báo cáo khoa học: Coiled–coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms pptx

Coiled–coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms ´ Rene E M A van Herpen, Jorrit V Tjeertes, Susan A M Mulders, Ralph J A Oude Ophuis, ´ Be Wieringa and Derick G Wansink Department of Cell Biology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, the Netherlands Keywords coiled-coil domain; multimerization; myotonic dystrophy protein kinase; protein– protein interaction; Rho kinase family Correspondence D G Wansink, Department of Cell Biology (code 283), NCMLS, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, the Netherlands Fax: +31 24 3615317 Tel: +31 24 3613664 ⁄ 14329 E-mail: r.wansink@ncmls.ru.nl Website: http://www.ncmls.nl (Received 24 November 2005, revised 11 January 2006, accepted 16 January 2006) doi:10.1111/j.1742-4658.2006.05138.x The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val– Ser–Gly–Gly–Gly motif Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization This multimerization is mediated by coiled–coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled–coil interaction was perturbed Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics Myotonic dystrophy protein kinase (DMPK) was first identified and cloned as a protein kinase in the quest to establish the molecular basis of disease in myotonic dystrophy, now more than a decade ago Study of the structure–function relationship of domains in DMPK and homologous kinases, such as the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKa ⁄ -bc) [1,2], ROCK-I ⁄ -II [3] and Citron kinase [4], placed DMPK in the large AGC group of protein kinases [5–7] MRCKs, ROCKs and Citron kinase regulate and reorganize the actin-based cytoskeleton as effectors of the small GTPases Cdc42 or Rho [8] Their kinase activity controls the status of myosin regulatory light chain phosphorylation, either directly or indirectly via regulation of myosin phosphatase activity, thereby affecting stress fiber formation, smooth muscle contraction or cytokinesis [9–11] Although myosin phosphatase targeting subunit (MYPT1) has been identified as a substrate for DMPK [5,12], the effects of DMPKmediated phosphorylation on actomyosin dynamics Abbreviations CM , coil mutant; DMPK, myotonic dystrophy protein kinase; DSP, dithiobis (succinimidyl propionate); ER, endoplasmic reticulum; MOM, mitochondrial outer membrane; MRCK, myotonic dystrophy kinase-related Cdc42-binding kinase; MYPT, myosin phosphatase targeting subunit 1124 FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al have not yet been studied in detail In addition, the role of other domains in DMPK, and their possible involvement in the regulation of catalytic kinase activity, has, to date, remained elusive Homology comparison can help, as members of the DMPK family have, to some degree, a similar domain arrangement [3,5,13] In all members of the DMPK family of protein kinases, a conserved leucine-rich N terminus of  70 amino acids precedes the catalytic kinase domain, which is followed by a characteristic coiled-coil region at the C-terminal end [1,4,14] In DMPK, next to these shared protein domains, two alternatively spliced domains were identified (a) a five-amino acid Val–Ser– Gly–Gly–Gly (VSGGG) sequence and (b) the DMPK C terminus [15] In vitro study of one of the six major DMPK splice isoforms has revealed that a relationship must exist between kinase activity and the state of multimerization promoted by the N terminus and the coiled-coil domain [16,17] However, the consequences of multimerization and association with other proteins for in vivo activity regulation of DMPK are not clear Activation of Citron kinase and ROCK-I is mediated by RhoA binding to a Rho-binding domain located in the C-terminal part of the coiled-coil region 1[4,8,19] In this respect, the coiled-coil region fulfils a regulatory role, as RhoA binding relieves inhibition imposed by the C terminus on the kinase domain [20] Furthermore, the coiled-coil segment seems to carry out a special role in regulating the multimeric state of ROCK-I and MRCKa, thereby regulating their kinase activity [14,20,21] Dimer formation mediated by the N terminus of MRCKa is followed by transautophosphorylation and also contributes to regulation of the MRCKa catalytic activity [14] For DMPK, it has been reported that myotonic dystrophy protein kinase-binding protein enhances DMPK catalytic activity [22] In addition, binding of the Rho-family member, Rac-1, and phosphorylation by Raf-1, serve as activating events [23] We have recently reported that the different alternative C termini anchor DMPK isoforms in distinct intracellular membranes, targeting DMPK isoforms A and B to the endoplasmic reticulum (ER) and DMPK C and D to the mitochondrial outer membrane (MOM) [5,24] Specific elements in the coiled-coil domain exclusively affect the mitochondrial but not the ER targeting behavior [24] Short DMPK isoforms E and F, containing a two-amino acid C terminus following the coiled-coil domain, adopt a cytosolic localization Furthermore, the VSGGG motif, unique among AGC kinases, regulates DMPK autophosphorylation, in-gel migration behavior and, probably, folding [5] To fully understand how the individual DMPK isoform structure relates to function, we focus here on DMPK coiled-coils mediate multimerization the significance of the coiled-coil domain in the regulation of multimerization, kinase activity and localization behavior Using biochemical and cell biological approaches, we demonstrate that the tendency of DMPK to multimerize to higher-molecular weight complexes relies on typical structural sequence properties of the coiled-coil segment, independent of kinase activity or the presence of other alternatively spliced domains Reciprocal effects were also seen because coiled–coil interactions modulated, but did not abolish, autophosphorylation ability, transphosphorylation activity towards peptide substrates in vitro, complex formation with the DMPK substrate MYPT2 and, in the case of DMPK C, localization to mitochondria in vivo Results Individual DMPK isoforms exist in high-molecularweight complexes DMPK isoforms (Fig 1) differ in localization, enzymatic activity and autophosphorylation, owing to alternatively spliced domains [5,24] As a first step towards a better understanding of the role of the coiled-coil segment in the differential structure–function properties of the different isoforms, we performed gel filtration experiments to obtain a size estimate of the complexes in which DMPK isoforms can reside Cleared lysates of cells expressing DMPK A, C, E or F were applied to a Superose gel-filtration column calibrated with standard molecular weight markers Elution profiles were traced with western blotting Full-length DMPK isoforms A and C (predicted molecular mass  70 kDa; apparent molecular mass  75 kDa on an SDS ⁄ PAGE gel) eluted as large complexes, with the main signal exceeding 440 kDa in molecular mass (Fig 2) Breakdown products of these large DMPKs, inevitably formed by in vitro product handling in the experimental procedures used, appeared predominantly in the same fractions as the BSA marker protein ( 67 kDa) Splice isoforms E and F (predicted molecular mass  60 kDa; apparent molecular mass  68 kDa on an SDS ⁄ PAGE gel) were also found in high-molecular-weight complexes, but did not yield any breakdown products because they lack the long C-terminal tail domains that confer proteolytic vulnerability (see Fig 1) [5,15] The elution profiles of isoforms E and F, which only differ in the presence of a VSGGG motif, were comparable, suggesting that the VSGGG motif has no role in complex formation (note the characteristic doublet signal for isoforms containing a VSGGG motif, which is related FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS 1125 DMPK coiled-coils mediate multimerization R E M A van Herpen et al Fig Myotonic dystrophy protein kinase (DMPK) isoforms and truncation mutants Structural domain organization of DMPK splice isoforms A, C, E and F, and N-terminal and C-terminal truncation mutants of DMPK E used in this study, are shown The N terminus, Ser ⁄ Thr protein kinase domain, alternatively spliced VSGGG motif, coiled-coil domain and alternatively spliced tail regions are indicated Truncation mutants contain an N-terminal VSV-tag Numbers refer to amino acid numbering in full-length DMPK E and indicate the first and last amino acid of the DMPK segment contained within the mutants The ability to multimerize is indicated (see the text) Fig Myotonic dystrophy protein kinase (DMPK) isoforms reside in high-molecular-weight complexes Size exclusion chromatography was performed on cell-free extracts of transfected COS-1 cells containing DMPK A, C, E, F or VSV-DMPK E(402–537) Fractions were analyzed on western blots using an anti-DMPK immunoglobulin Molecular mass markers ribonuclease A (14 kDa), BSA (67 kDa), aldolase (150 kDa) and ferritin (440 kDa) were used to calibrate the fraction volume positions of differently sized proteins in the column eluate (indicated on top) Molecular mass markers for SDS ⁄ PAGE are indicated at the left (30, 65 and 83 kDa) All fulllength isoforms and also truncation mutant VSV-DMPK E(402–537) were found in large multimeric complexes During the procedure, loss of the C terminus occurred for isoforms A and C, which has been described previously [5,15] to autophosphorylation) [5] Taken together, these experiments reveal that all individual DMPK isoforms occur in large, multimeric complexes The coiled-coil domain mediates DMPK multimerization To investigate whether multimerization is involved in the formation of large complexes, and to identify the protein domains involved, we introduced various N-terminal and C-terminal truncation mutations into 1126 DMPK isoform E (Fig 1) COS-1 cells were doubly transfected with full-length HA-DMPK E and one VSV-tagged truncation mutant, and extracts were tested in co-immunoprecipitation experiments (Fig 3A) HA-DMPK E did not precipitate the N-terminal region fused to the kinase domain, irrespective of the presence of the VSGGG motif Similarly, the kinase domain alone, VSV-DMPK E(60–375), did not interact with full-length HA-DMPK E In contrast, the C-terminal region of DMPK E, containing the coiledcoil domain, with or without the VSGGG motif [i.e constructs E(340–537) and E(402–537)], did associate with HA-DMPK E The specificity of this interaction was also observed in a reverse immunoprecipitation using the anti-VSV immunoglobulin (Fig 3B) Independently, gel filtration confirmed that VSV-DMPK E(402–537) participated in high-molecular-weight complexes (Fig 2) Together, our findings suggest that only the coiled-coil domain is relevant for interaction DMPK homo- and heteromultimerization occurs independently of kinase activity and alternatively spliced domains To confirm that DMPK self-association indeed occurred independently of kinase activity, a kinase dead mutant was tested in a co-immunoprecipitation experiment As evident from the results shown in Fig 4A, both truncation mutants containing the coiled-coil domain coprecipitated with HA-DMPK E(K100A), a kinase-inactive variant impaired in ATP binding owing to a lysine to alanine mutation in the kinase domain [5] DMPK splice isoforms are expressed in a cell-typespecific manner The main isoforms in skeletal muscle, FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al DMPK coiled-coils mediate multimerization A B * Fig The coiled-coil domain mediates self-association of myotonic dystrophy protein kinase (DMPK) E (A) Full-length HA-DMPK E was co-expressed with VSV-tagged DMPK truncation mutants in COS-1 cells, as indicated on top (+, present; ), not present) Immunoprecipitations (IP) were carried out using anti-HA-coated beads DMPK E interacted only with mutants containing a coiled-coil domain [i.e E(340–537) and E(402–537)] (B) The coiled–coil mediated interaction was confirmed by reverse IPs with anti-VSV on lysates containing untagged DMPK E and VSV-DMPK E(340–537) or VSV-E(402–537) Precipitated proteins were detected on western blots with anti-HA, anti-VSV or anti-DMPK, as indicated The asterisk indicates a nonspecific signal detected by the anti-HA immunoglobulin in whole cell lysates A B C * Fig Multimerization of myotonic dystrophy protein kinase (DMPK) is independent of kinase activity or alternatively spliced domains (A) Involvement of kinase activity in DMPK complex formation was investigated by immunoprecipitation (IP) using lysates expressing the DMPK inactive mutant E(K100A) and truncation mutants VSV-DMPK E(340–537) or E(402–537) as indicated on top (+, present; ), not present) (B) To investigate the effects of the VSGGG motif on multimerization, HA- and His-tagged versions of DMPK E and F were used in IPs, as indicated The asterisk indicates a nonspecific signal detected by the anti-HA immunoglobulin in whole cell lysates (C) Involvement of the C terminus in multimerization was examined by the expression of combinations of YFP–DMPK A or C and His-DMPK C or E, as indicated IPs were performed using anti-HA or anti-YFP, and western blots were probed with anti-DMPK, anti-His, anti-HA or anti-YFP, as indicated heart and brain are DMPK A–D, whereas DMPK E and F predominate in smooth muscle tissue [15] It was previously shown that the VSGGG motif in DMPK A, C and E enhances autophosphorylation [5] To study a potential modulatory effect of the VSGGG motif on DMPK self-association, the interaction between DMPK isoforms E (includes a VSGGG sequence) and F (no VSGGG sequence) was examined Again, when His tags and HA tags were used to discriminate between the different splice isoform partners, we observed homomultimerization by DMPK isoforms E and F, as well as heteromultimerization between isoforms E and F (Fig 4B) To investigate whether different tail regions affected DMPK self-association (Fig 1), we tested isoform combinations A with C, A with E, and C with E, and again found all of these iso- FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS 1127 DMPK coiled-coils mediate multimerization R E M A van Herpen et al forms to interact independently of their C-terminal sequence (Fig 4C; data not shown) Taken together, these results provide strong evidence that DMPK multimerization is an intrinsic property of all DMPK isoforms, and that homomultimers as well as heteromultimers can be formed when the 402–537 region is present Mutations in the coiled-coil region impair DMPK multimerization We used coils, paircoil and multicoil algorithms to compute the probability of coiled-coil formation across the stretch of amino acids between positions 340 through 537 in DMPK E [25–27] Nine heptad-repeat sequences were identified by these algorithms, predicting a coiled-coil probability of almost for the entire segment between amino acids 469 and 531 (Fig 5A) Combined with the knowledge that 3.5 amino acids are needed to complete one turn in a coil, this suggests that 18 turns make up the entire DMPK a-helical coil [28] At the a and d positions of every heptad, known to make up the typical hydrophobic interface between interacting coiled-coil domains [28], over 50% of the residues are of hydrophobic nonaromatic nature (i.e leucine, isoleucine or valine) (Fig 5B) Lysine residues, which are commonly found at electrostatic residue positions e and g in the heptad, are absent in the DMPK coiled coil Instead, arginines were found at these positions in heptads IV, VI and VIII Database comparison of the DMPK coiled-coil region showed homology to distinct parts of the large coiled-coil region of myosin heavy chain, MRCKa ⁄ -b ⁄ -c and ROCK-I ⁄ -II ( 25% identity;  60% similarity) [5] In order to approach the anticipated structural role of the coil experimentally, we mutated amino acid residues at the a and d positions within heptad repeats II, III and VII, because these are known to be of crucial importance for coiled-coil formation Leucine to glycine changes at positions 477, 487 and 515 were introduced, because, according to the paircoil algorithm, these would lower the coiled-coil probability to < 0.5 (Fig 5A) Transfection in COS-1 cells showed that expression levels of the HA-DMPK E mutant with point mutations L477G, L487G and L515G [hereafter designated HA-DMPK E coil mutant (HA-DMPK ECM)] and HA-DMPK E were similar (Fig 6A) HA-DMPK ECM migrated more slowly in the gel, indicating that its protein conformation had indeed changed as a result of the Leu to Gly mutations This altered migration was specifically caused by the L477G mutation, as the two other mutations did not contribute to the effect (data not shown) Immunoprecipitation of HA-DMPK ECM was less efficient than of HA-DMPK E When using a stepwise increasing series of antiserum concentrations, the amount of precipitated HA-DMPK ECM was consistently lower than that of HA-DMPK E, indicating impaired self-association of HA-DMPK ECM (Fig 6B) Co-immunoprecipitation with differentially tagged DMPK E confirmed that DMPK ECM could not engage in homodimerization This was confirmed when HA-DMPK ECM was unable to associate with and pull down full-length His-DMPK E under conditions of excess anti-HA beads (Fig 6C) Furthermore, the association of DMPK ECM with truncation mutants DMPK E(340–537) and E(402–537) was not observed (data not shown), indicating that leucines 477, 487 and Fig Prediction of coiled-coil probability and heptad repeats in myotonic dystrophy protein kinase (DMPK) (A) Computational analysis of the coiled-coil forming probability of the DMPK segment spanning amino acids 340–537 using the programs COIL, PAIRCOIL and MULTICOIL When leucine at positions 477, 487 and 515 were replaced by glycine, as in the DMPK E coil mutant (DMPK ECM), the predicted coil between amino acids 469–531 dropped below 50% probability (PAIRCOIL) (B) Assignment of a heptad repeat register (a–g) for amino acids 469–531 of DMPK based on predictions made by COIL, PAIRCOIL and MULTICOIL The DMPK coiled-coil domain contains nine heptad repeats, indicated by Roman numerals Leucine to glycine mutations in DMPK ECM are boxed 1128 FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al DMPK coiled-coils mediate multimerization Fig Coiled-coil mutations in myotonic dystrophy protein kinase (DMPK) E impair self-association (A) Expression of the HA-DMPK E coil mutant (HA-DMPK ECM) in COS-1 cells was examined by western blotting using an anti-HA immunoglobulin Using HA-DMPK E as a reference, HA-DMPK ECM displayed an altered gel mobility (+, present; ), not present) The upper band of the HA-DMPK ECM doublet co-migrated with a nonspecific signal (marked with an asterisk) (B) Immunoprecipitations on extracts containing HA-DMPK E or HA-DMPK ECM were carried out with a series of increasing concentrations of anti-HA immunoglobulin DMPK products in cell lysates (CL) and immunoprecipitates (IP) were probed with anti-HA on western blots Loading was 1% of total for the cell lysate and 2% of total for the immunoprecipitates Brackets indicate positions of heavy chains of the anti-HA immunoglobulin The graph shows the immunoprecipitation efficiency for each protein, determined by densitometrical scanning of the DMPK signal and plotted against the concentration of anti-HA immunoglobulin used in the immunoprecipitation (C) Immunoprecipitations were performed on lysates containing His-DMPK E together with either HA-DMPK E or HA-DMPK ECM (+, present; ), not present) DMPK proteins were probed with an anti-HA or anti-His immunoglobulin on a western blot 515 within the coiled-coil domain are essential for the self-association behavior of DMPK E Coiled-coil mutations reduce, but not abolish, DMPK kinase activity Earlier work of our group and others has demonstrated that DMPK E phosphorylates MYPT1 [5] (data not shown) and thereby inhibits myosin phosphatase activity [12] We tested here whether impaired coiled– coil interactions would affect DMPK substrate binding and kinase activity MYPT2, a paralogue of MYPT1 [29] was co-expressed with DMPK E or ECM in COS-1 cells and their interaction analyzed by immunoprecipitation A small, but significant, fraction of MYPT2 was complexed to DMPK E, but no interaction could be detected with the coil mutant (Fig 7A) Owing to incomplete reduction of the reversible chemical crosslinker dithiobis (succinimidyl propionate) (DSP), used to stabilize the DMPK–MYPT2 interaction and only included for DMPK–MYPT2 co-expression studies, a considerable fraction of DMPK E complexes migrated as high-molecular-weight structures in the gel (asterisks in Fig 7A) This was also observed for lysates that contained DMPK ECM, albeit at a lower signal intensity The most simple explanation for the latter obser- vation is that, despite the perturbed coiled-coil domain, other parts of the protein could still be involved in association behavior We next examined, in an in vitro kinase assay, whether the association (i.e as assessed by immunoprecipitation) between DMPK and MYPT2 was a prerequisite for MYPT2 phosphorylation Much to our surprise, MYPT2 was phosphorylated by DMPK E and DMPK ECM at almost similar efficiency (Fig 7B) In addition, autophosphorylation was still present in DMPK ECM, but this was two- to threefold lower than in wild-type DMPK E (Fig 7B) More quantitatively, we examined how coil mutations affected kinase activity in an assay based on the preferred DMPK peptide substrate, KKRNRRLTVA [5] Under these conditions, both peptide phosphorylation and autophosphorylation by DMPK ECM was approximately threefold lower than peptide phosphorylation and autophosphorylation by DMPK E (Fig 7C) By examination of steady-state levels and structural intactness of DMPK protein products, we ruled out that altered proteolytic processing was underlying this effect (data not shown) Combined, these results thus suggest that the coil region must have a facilitating, rather than an essential, role in the determination of DMPK activity and specificity FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS 1129 DMPK coiled-coils mediate multimerization R E M A van Herpen et al Fig The myotonic dystrophy protein kinase E coil mutant (DMPK ECM) displays reduced kinase activity (A) Myosin phosphatase targeting subunit (MYPT2) interaction with DMPK E depends on an intact coiled-coil domain HA-DMPK E or ECM were co-expressed with VSV-MYPT2 in COS-1 cells (+, present; ), not present) Lysates were prepared in the presence of the cross-linker, dithiobis (succinimidyl propionate), and used in immunoprecipitations with anti-HA beads Proteins in the cell lysate (input) and after immunoprecipitation (IP) were probed on a western blot with anti-DMPK or anti-MYPT immunoglobulin The asterisk marks slow-migrating complexes resistant to decrosslinking, which contain DMPK (B) DMPK ECM showed reduced autophosphorylation, but was capable of phosphorylating MYPT2 Immunopurified MYPT2 was used in a kinase assay with purified HA-DMPK E, ECM or E(K100A) (+, present; ), not present) Western blotting was used to validate the input of DMPKs and MYPT Phosphorylation of MYPT2 and autophosphorylation of DMPK were visualized by autoradiography and quantified by phosphoimager analysis The MYPT2 background signal, caused by copurifying kinase activity and nearly equal to the signal observed using HADMPK E(K100A), was subtracted [5] (C) DMPK ECM displayed reduced kinase activity towards a peptide substrate Immunopurified HA-DMPK E, ECM and E(K100A) were used in a kinase assay with preferred peptide substrate, KKRNRRLTVA [5] (+, present; ), not present) Input of DMPK in the assay was validated by western blotting The coiled-coil domain stabilizes DMPK C interaction with mitochondria Immediately C-terminal to the coiled-coil segment, spaced by a stretch of only five amino acids, is the alternatively spliced DMPK C terminus, which determines subcellular targeting to the ER (mDMPK A), the MOM (mDMPK C), or the cytosol (DMPK E) [5] We have reported that the presence of the coiled-coil region in DMPK C is essential for MOM anchoring, but it remained unclear whether this effect should be 1130 attributed to structural integrity of the entire coiledcoil domain, or to properties of any particular amino acid segment therein [24] We compared the localization of YFP-tagged isoforms A, C and E, containing the L477G, L487G and L515G mutations, with that of the wild type YFP–DMPK isoforms (Fig 8) As shown in Fig 8A–F, YFP–DMPK CCM was partitioned over MOM and the cytosol, whereas nonmutated YFP–DMPK C was located uniquely at mitochondria To us this suggests that coiled coilmediated associations contribute to DMPK–MOM FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al DMPK coiled-coils mediate multimerization A B C D E F G H I Fig Myotonic dystrophy protein kinase (DMPK) C association with mitochondria is stabilized by the coiled-coil domain (A–C) YFP–DMPK C colocalized with mitochondria stained with a cytochrome c oxidase antibody (D–F) The YFP–DMPK C coil mutant (YFP–DMPK CCM) was not only located at mitochondria but was also found dispersed throughout the cytosol (G) The cytosolic distribution of YFP–DMPK ECM is not altered by introduction of the mutations L477G, L487G and L515G (see YFP–DMPK E in the insert) (H–I) Expression of YFP–DMPK A and ACM in N2A cells showed identical endoplasmic reticulum (ER) localizations for both proteins Bars, 10 lm binding strength, but are not essential Although considered less likely, an alternative explanation could be that abnormal properties of the coiled-coil structure render the tail in DMPK C less avid to engage in MOM binding We also examined whether the coil mutations had any effect on the distribution of DMPK A (present in the ER membrane) and DMPK E (cytosolic variant) Figure 8G–I show that the locations of the DMPK ACM and ECM remained unchanged, corroborating previous findings that unique properties of the A and C tails drive localization [24] Discussion The results presented here provide evidence for the contention that the intact coiled-coil region, presuma- bly by means of its unique helical ⁄ structural properties, is a key factor in aggregation behavior and a modifier of biological properties of the adjacent domains (i.e the kinase and tail domains) in DMPK The coiled-coil region thus codetermines the unique structure–function characteristics of each of the six major DMPK isoforms The coiled-coil domain mediates DMPK homo- and heteromultimerization Sizing experiments with gel filtration chromatography revealed that full-length DMPK isoforms reside in high-molecular-weight multimeric complexes Pure DMPK dimers may exist, but form only a minor fraction of total protein Given the elution profile, and FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS 1131 DMPK coiled-coils mediate multimerization R E M A van Herpen et al based on an average complex size of 0.5 MDa and a molecular mass for individual isoforms of 60–70 kDa, we estimate that the DMPK complex must contain around six monomers Fewer monomers may be possible if a considerable fraction of the complex is made up of different DMPK interacting proteins (i.e other than DMPK itself) Conservation of multimerization capacity in truncation mutant DMPK E(402–537), and impaired self-association of DMPK ECM, provide strong experimental evidence that the coiled-coil sequence is uniquely responsible for complex formation Moreover, our data rule out dominant involvement of the N-terminal region, kinase domain and amino acids 402–468 flanking the coiled-coil domain, including the alternatively spliced VSGGG motif or membrane anchors in the C-tail region The tendency to multimerize via coiled–coil association, but the apparent lack of effect of other protein motifs present in the protein, distinguishes DMPK from some of its closest relatives The N-terminal region of MRCKa mediates dimerization of the kinase domain independently of the coiled-coil domain [14] In the case of ROCK-II, removal of the large coiledcoil domain still results in the presence of a dimeric protein Here, dimerization may be driven by a small coiled-coil region in the N-terminal region [30] Although homology exists among the N termini of DMPK, ROCK-I ⁄ -II and MRCKa ⁄ -b ⁄ -c (i.e a leucine zipper-like motif is found in all), we consider it unlikely that the N terminus has a strong role in DMPK self-association, as mutant DMPK E(1–375) did not multimerize under the experimental conditions used A supporting role for the N terminus cannot be completely ruled out, however To provide evidence that it is the typical 3D coiledcoil organization and not the linear peptide sequence of the segment that is important for DMPK complex formation, we mutated three hydrophobic residues at the putative heptad positions a and d, known to stabilize the hydrophobic interface between helices forming the coiled coil [31,32] These mutations strongly influenced the in-gel-migration behavior of DMPK, and the single mutation L477G had already resulted in a remarkable migration shift, indicative of structural alterations introduced within the coiled-coil domain Most likely, the introduced glycine residues break up the helical coiled-coil conformation [33], whereas the hydrophobicity changes alter the folding behavior within the coil [28] From our experiments, it became clear that mutated positions a and d strongly reduced the self-association of DMPK E, providing evidence that it is the coiled-coil structure proper that deter1132 mines the self-association tendency Although residual self-association of DMPK ECM could be demonstrated through covalent cross-linking, we conclude that this mutant provides a proficient tool to study multimerization-related functions of DMPK Multimerization modulates DMPK kinase activity, substrate binding and localization We observed that DMPK ECM autophosphorylation and the transphosphorylation activity towards a peptide substrate was two to threefold reduced when compared with the corresponding activities of wild-type DMPK E To us this suggests that DMPK autophosphorylation is largely an intermolecular reaction in a homo- or heteromultimeric complex of DMPK isoforms We cannot rule out, however, the alternative possibility that distortion of the coiled-coil structure affects conformational flexibility in the kinase domain itself and that this feature is needed for efficient intramolecular autophosphorylation More detailed understanding of the DMPK structure is needed to be able to distinguish between these possibilities In DMPKlike kinases, multimerization capacity is apparently a prerequisite for proper kinase activation: the kinase activity of Rho-kinase and MRCKa is also partly dependent on the presence of a coiled-coil domain [14,18,20] and others have shown that multimerization of the human DMPK A isoform is correlated with increased activity [16,17] In contrast to the findings discussed above, we found that DMPK ECM was able to phosphorylate its natural substrate, MYPT2 Our inability to detect effects of the coil mutation on MYPT2 phosphorylation may be a result of the experimental conditions used In our assay system, only a limited amount of  0.2 lm MYPT2 was present, in contrast to the excess of 30 lm peptide used in the peptide kinase assay [5] However, because we were not able to stabilize the DMPK ECM MYPT2 interaction with a chemical cross-linker (Fig 7A), and the amount of MYPT2 bound to DMPK ECM was clearly lower than with wild-type DMPK E, we assume that the DMPK ECM MYPT2 binding is short-lived The DMPK coiled-coil domain may be important in strengthening the binding between DMPK and MYPT2 or in bringing protein sequences, crucial for cross-link formation, in close proximity Whether it is the coiled-coil region in MYPT2 that plays a role in the DMPK MYPT interaction will be investigated in future studies [5] (D G Wansink et al unpublished results) ER or MOM targeting of full-length mouse DMPK A and C, respectively, critically depends on the final 45 FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al C-terminal amino acids [24] Here, we show that mutational disruption of the structural integrity of the coiled-coil segment relocalizes DMPK C – but not DMPK A – to the cytosol One explanation for this observation would be that loss of multimerization causes increased exposure of YFP–DMPK C to the proteolytic machinery In turn, clipping could be associated with increased release of the YFP reporter moiety into the cytosol Western blotting and analysis of YFP– DMPK products in our transfected cell lines, however, ruled out this possibility (data not shown) Evidently, structural intactness of the coiled-coil domain stabilizes DMPK C association to the MOM, but is less critical for ER association of DMPK A It has been reported that formation of a coiled-coil structure utilizes a twostep model where protein folding and multimerization are coupled events [28] If this model holds, and given the fact that the membrane anchors are situated at the very C-terminal end of newly produced DMPK polypeptide chains, this would make it likely that DMPK anchoring into ER or mitochondrial membranes occurs when isoforms are already in a multimeric state Thus, multimerization per se may have co-operative effects and promote MOM binding, but, based on the evidence provided, we cannot exclude the possibility that the coiled-coil mutations perturb proper DMPK C-tail conformation, thereby reducing the membrane-anchoring affinity of individual polypeptide chains Currently, not enough is known about the molecular events involved in targeting tail-anchored proteins to mitochondria [34] to discriminate between these possibilities It is evident that the DMPK coiled-coil domain itself has no targeting properties [24]; however, it remains possible that, once targeted to mitochondria, the coiled-coil domain has some affinity for the mitochondrial membrane, as recently demonstrated for the coiled-coil domain in mitochondrial targeting of DLP1 ⁄ Drp1 [35] Taken together, our results provide evidence that the coiled-coil domain is crucial for homo- and heteromultimerization of DMPK isoforms and that multimerization has a function in substrate binding, phosphorylation and subcellular targeting properties of individual DMPK isoforms Whether DMPK complex formation is actively regulated in vivo (e.g to modulate DMPK activity and downstream effects), remains to be investigated Experimental procedures Cell culture and transfection Neuro-2A (N2A) and COS-1 cells were cultured and transfected as described previously [24] DMPK coiled-coils mediate multimerization Expression plasmids and site-directed mutagenesis Expression vectors for HA-, His- and EYFP-tagged DMPK A–F have been described previously [5,24] Expression plasmids, encoding VSV-tagged DMPK truncation constructs, were obtained by cloning PCR fragments amplified from template pSGmDMPK E [15] with the use of Pfu polymerase and specific primers EcoRI and XhoI sites were incorporated in the forward and reverse primers, respectively (underlined, see below) DNA fragments were cut with EcoRI and XhoI, gel purified and ligated into EcoRI and XhoI polylinker sites of plasmid pSG8VSV The sequence of all PCR fragments was verified by DNA sequencing The following primers were used: pSGVSVDMPK E(1–375): 5¢-ATAGAATTCATGTCAGCCGAAGTGCG3¢ and 5¢-ATTCTCGAGTCAAGTGAGCCGGTCCTCCA3¢; pSGVSVDMPK E(1–400): 5¢-ATAGAATTCATGTCA GCCGAAGTGCG-3¢ and 5¢-AATCTCGAGTCAGAAGG GCAGGCGCAC-3¢; pSGVSVDMPK E(60–375): 5¢-ATA GAATTCAGGCTTAAGGAGGTCCGA-3¢ and 5¢-ATT CTCGAGTCAAGTGAGCCGGTCCTCCA-3¢; pSGVSVD MPK E(340–537): 5¢-ATTGAATTCTTTGGCCTTGATTG GGA-3¢ and 5¢-ATACTCGAGCTAGGGATCTGCGGCT3¢; pSGVSVDMPK E(402–537): 5¢-ATAGAATTCGGCTA CTCCTACTGCTGCAT-3¢ and 5¢-ATACTCGAGCTAGG GATCTGCGGCT-3¢ To generate the HA-DMPK E coil mutant expression vector, pSGHADMPK ECM, three amino acid mutations were introduced into full-length pSGHADMPK E with use of the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), according to the manufacturer’s protocol, in successive mutagenesis steps The following primers were used: for L477G, primers 5¢-CAGCTCCAGG AAGCCGGGGAAGAAGAGGTTC-3¢ and 5¢-GAACCT CTTCTTCCCCGGCTTCCTGGAGCTG-3¢; for L487G, primers 5¢-TCACCCGGCAGAGCGGGAGCCGCGAGC TGGAG-3¢ and 5¢-CTCCAGCTCGCGGCTCCCGCTCTG CCGGGTGA-3¢; for L515G, primers 5¢-GTCCGAAACC GAGACGGGGAGGCGCATGTTC-3¢ and 5¢-GAACATG CGCCTCCCCGTCTCGGTTTCGGAC-3¢ To generate expression plasmids pEYFP-DMPK ACM, CM and ECM, the following cloning steps were carried out C First, an L515G mutation was introduced into pSGHADMPK A and C, as described above, resulting in pSGHADMPK A(L515G) and C(L515G) Then, two fragments [an AflII–BspEI fragment – common to all DMPK isoforms and including mutations L477G and L487G – isolated from pSGHADMPK ECM, and a BspEI–BsrGI fragment – specific for each individual DMPK isoform, and including mutation L515G isolated from pSGHADMPK A(L515G), C(L515G) and ECM] were ligated into a pSGmDMPK E vector digested with AflII and BsrGI, resulting in pSGDMPK ACM, CCM and ECM Finally, BglII-flanked cDNAs from pSGHADMPK ACM, CCM and FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS 1133 DMPK coiled-coils mediate multimerization R E M A van Herpen et al ECM were ligated into the BglII site of plasmid pEYFP-C1 (Clontech, Mountain View, CA, USA) MYPT2 was cloned by RT-PCR using mouse skeletal muscle RNA and primers 5¢-ATAGAATTCATGGCGGA GCTGGAGCA-3¢ and 5¢-ATACTCGAGCTACTTGGAC AGTTTGCTGATGACT-3¢ (start and stop codons underlined) in a pSG8-based vector in-frame with a His and a VSV tag sequence Gel filtration chromatography Expression plasmids pSGDMPK A, C, E, F or pSGVSVDMPK E(402–537) were transfected into COS-1 cells grown on 10 cm dishes and cultured for an additional 24 h prior to analysis Then, cells were collected and the cleared cell extracts were prepared by centrifugation after lysis of cells in RIPA buffer [50 mm Hepes, pH 7.5, 150 mm NaCl, mm EDTA, 25 mm NaF, 1% (w ⁄ v) Triton X-100, 1% (w ⁄ v) sodium desoxycholate, 0.1% (w ⁄ v) SDS, mm phenylmethanesulfonyl fluoride) on ice Gel filtration was performed on a SMART system Superose HR10 ⁄ 30 column (GE Healthcare, Roosendaal, the Netherlands) equilibrated with RIPA buffer and calibrated with molecular mass markers ferritin (440 kDa), alcohol dehydrogenase (150 kDa), BSA (67 kDa) and ribonuclease A (14 kDa) Cell extracts (10 lL) were applied to the column at a flow rate of 40 lLỈmin)1 Fractions of 40 lL were collected and the protein content in each fraction was subjected to SDS ⁄ PAGE analysis Confocal microscopy N2A cells were grown on glass coverslips and transfected with expression plasmids pEYFP-DMPK A, C, E, ACM, CCM or ECM After 24 h, cells were fixed in NaCl ⁄ Pi (PBS), containing 2% (w ⁄ v) formaldehyde, and either mounted directly or processed for immunofluorescence using standard procedures [5] A rabbit anti-(cytochrome c) oxidase immunoglobulin was used to visualize mitochondria Confocal images were generated on a Bio-Rad MRC1024 confocal laser-scanning microscope (Bio-Rad, Hercules, CA, USA) equipped with an argon ⁄ krypton laser, using a 60 · 1.4 NA oil objective and lasersharp2000 acquisition software Images were further processed with adobe photoshop 7.0 (Adobe, San Jose, CA, USA) 0.1 mm Na3VO4, lm Microcystin LR (ALEXIS, Lausen, Switzerland), mm phenylmethanesulfonyl fluoride, supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany)] The reversible chemical cross-linker, DSP (0.5 mgỈmL)1), was added to the lysis buffer in DMPK– MYPT2 co-expression studies only Co-immunoprecipitations were performed with an anti-HA (12CA5) or an antiVSV monoclonal antibody coupled to protein A–Sepharose beads Immunoprecipitates were washed four times in RIPA buffer, the final wash was removed and beads were mixed with Laemmli sample buffer and used for SDS ⁄ PAGE and western blot analysis on poly(vinylidene difluoride) membrane, as detailed below Kinase assays were performed as described previously [5] Briefly, beads derived from the immunoprecipitation were washed and aliquoted into the desired number of kinase reactions The final wash was removed and 30 lm peptide substrate KKRNRRLTVA or lg of immunopurified His-VSV-MYPT2 in kinase assay buffer was added [5] The reaction was started by the addition of [32P]ATP[cP] ( lCi) and the mixture was incubated at 30 °C (60 for the peptide, 20 for MYPT2) Phosphate incorporation into the peptide substrate under the conditions used was linear, and was analyzed as described previously [5] SDS ⁄ PAGE and western blotting Proteins were separated by SDS ⁄ PAGE, transfered to poly(vinylidene difluoride) membrane (GE Healthcare), then analyzed by immunodetection For visualization of DMPK, DMPK-specific antibody (B79) or anti-HA immunoglobulin was used [5,15]; truncation mutants were detected using an anti-VSV immunoglobulin Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Soham, UK) were used followed by enhanced chemiluminescence (ECL) and exposure to film (Kodak X-OMAT AR; Kodak, Paris, France) Bioinformatics Computational algorithms used to predict heptad sequences in DMPK included the program coil (v2.1, mtidk matrix and window 21) [26], paircoil (probability cut-off 0.5) [27] and multicoil (cut-off 0.5 and window 28) [25] Immunoprecipitation and in vitro kinase assay COS-1 cells were transfected with DMPK and ⁄ or MYPT2 expression plasmids, or with empty vector DNA (pSG8DEco), cultured for  24 h, washed with ice-cold PBS and lysed on ice in RIPA buffer or kinase assay lysis buffer [50 mm Tris ⁄ HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 25 mm NaF, mm sodium pyrophosphate, 1134 Acknowledgements We would like to express our gratitude to Dr J Schalkwijk (Department of Dermatology, RUMC Nijmegen) for advice on and help with gel-filtration chromatography This study was supported by the FEBS Journal 273 (2006) 1124–1136 ª 2006 The Authors Journal compilation ª 2006 FEBS R E M A van Herpen et al Prinses Beatrix 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Herpen et al Fig Myotonic dystrophy protein kinase (DMPK) isoforms and truncation mutants Structural domain organization of DMPK splice isoforms A, C, E and F, and N-terminal and C-terminal truncation... A B C * Fig Multimerization of myotonic dystrophy protein kinase (DMPK) is independent of kinase activity or alternatively spliced domains (A) Involvement of kinase activity in DMPK complex formation... regulation of the MRCKa catalytic activity [14] For DMPK, it has been reported that myotonic dystrophy protein kinase -binding protein enhances DMPK catalytic activity [22] In addition, binding of the