Although myosin phospha-tase targeting subunit 1 MYPT1 has been identified as a substrate for DMPK [5,12], the effects of DMPK-mediated phosphorylation on actomyosin dynamics Keywords coi
Trang 1mitochondrial 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
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
dystro-phy kinase-related Cdc42-binding kinase (MRCKa⁄
-b-c) [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 contrac-tion or cytokinesis [9–11] Although myosin phospha-tase targeting subunit 1 (MYPT1) has been identified as
a substrate for DMPK [5,12], the effects of DMPK-mediated phosphorylation on actomyosin dynamics
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 independ-ently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity Complex formation was impaired in myotonic dys-trophy 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 transphosphoryla-tion 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 pre-served, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced Further-more, 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
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.
Trang 2have not yet been studied in detail In addition, the role
of other domains in DMPK, and their possible
involve-ment 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
kin-ases, 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
cata-lytic 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
phos-phorylation by Raf-1, serve as activating events [23]
We have recently reported that the different
alternat-ive C termini anchor DMPK isoforms in distinct
intra-cellular 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
iso-form structure relates to function, we focus here on
the significance of the coiled-coil domain in the regula-tion of multimerizaregula-tion, kinase activity and localiza-tion 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 proper-ties 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-molecular-weight complexes
DMPK isoforms (Fig 1) differ in localization, enzy-matic 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–func-tion 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 com-plexes, 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 pro-teolytic 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, sug-gesting that the VSGGG motif has no role in complex formation (note the characteristic doublet signal for isoforms containing a VSGGG motif, which is related
Trang 3to 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
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 tes-ted 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 inter-act with full-length HA-DMPK E In contrast, the C-terminal region of DMPK E, containing the coiled-coil 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) Inde-pendently, gel filtration confirmed that VSV-DMPK E(402–537) participated in high-molecular-weight com-plexes (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 trunca-tion mutants containing the coiled-coil domain copre-cipitated 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-type-specific manner The main isoforms in skeletal muscle,
Fig 2 Myotonic dystrophy protein kinase (DMPK) isoforms reside
in high-molecular-weight complexes Size exclusion
chromatogra-phy 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
immuno-globulin Molecular mass markers ribonuclease A (14 kDa), BSA
(67 kDa), aldolase (150 kDa) and ferritin (440 kDa) were used to
cal-ibrate 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
full-length 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].
Fig 1 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).
Trang 4heart 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 dis-criminate 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
*
Fig 3 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) Immunoprecipita-tions (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.
*
Fig 4 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.
Trang 5forms to interact independently of their C-terminal
sequence (Fig 4C; data not shown) Taken together,
these results provide strong evidence that DMPK
mul-timerization is an intrinsic property of all DMPK
isoforms, and that homomultimers as well as
hetero-multimers 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,
predict-ing a coiled-coil probability of almost 1 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 resi-dues 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 gly-cine changes at positions 477, 487 and 515 were intro-duced, 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 contrib-ute 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 consis-tently lower than that of HA-DMPK E, indicating im-paired 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 ECMwas 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 5 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 E CM ), 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 E CM are boxed.
Trang 6515 within the coiled-coil domain are essential for the
self-association behavior of DMPK E
Coiled-coil mutations reduce, but do not abolish,
DMPK kinase activity
Earlier work of our group and others has
demonstra-ted 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 ECMin COS-1
cells and their interaction analyzed by
immunoprecipi-tation 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
cross-linker 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
inten-sity 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, whe-ther the association (i.e as assessed by immunoprecipi-tation) 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 affec-ted kinase activity in an assay based on the preferred DMPK peptide substrate, KKRNRRLTVA [5] Under these conditions, both peptide phosphorylation and au-tophosphorylation by DMPK ECM was approximately threefold lower than peptide phosphorylation and au-tophosphorylation by DMPK E (Fig 7C) By exam-ination 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
Fig 6 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 refer-ence, HA-DMPK E CM displayed an altered gel mobility (+, present; ), not present) The upper band of the HA-DMPK E CM doublet co-migra-ted with a nonspecific signal (marked with an asterisk) (B) Immunoprecipitations on extracts containing HA-DMPK E or HA-DMPK E CM 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 E CM (+, present; ), not present) DMPK proteins were probed with an anti-HA or anti-His immunoglobulin on a western blot.
Trang 7The 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
deter-mines 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
attributed to structural integrity of the entire coiled-coil domain, or to properties of any particular amino acid segment therein [24] We compared the localiza-tion 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 parti-tioned over MOM and the cytosol, whereas
nonmutat-ed YFP–DMPK C was located uniquely at mitochondria To us this suggests that coiled coil-mediated associations contribute to DMPK–MOM
Fig 7 The myotonic dystrophy protein kinase E coil mutant (DMPK ECM) displays reduced kinase activity (A) Myosin phosphatase targeting subunit 2 (MYPT2) interaction with DMPK E depends on an intact coiled-coil domain HA-DMPK E or E CM 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 E CM showed reduced autophosphorylation, but was capable of phosphorylating MYPT2 Immunopurified MYPT2 was used in
a kinase assay with purified HA-DMPK E, ECMor 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 phos-phoimager analysis The MYPT2 background signal, caused by copurifying kinase activity and nearly equal to the signal observed using HA-DMPK E(K100A), was subtracted [5] (C) HA-DMPK ECMdisplayed reduced kinase activity towards a peptide substrate Immunopurified HA-DMPK
E, E CM 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.
Trang 8binding strength, but are not essential Although
con-sidered 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
(cyto-solic variant) Figure 8G–I show that the locations of
the DMPK ACMand ECMremained unchanged,
corro-borating 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 proper-ties, 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 frac-tion of total protein Given the elufrac-tion profile, and
Fig 8 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 E CM 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 ACMin N2A cells showed identical endoplasmic reticulum (ER) localizations for both proteins Bars, 10 lm.
Trang 9based 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
pos-sible 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
trunca-tion mutant DMPK E(402–537), and impaired
self-association of DMPK ECM, provide strong
experi-mental evidence that the coiled-coil sequence is
uniquely responsible for complex formation
More-over, 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
associ-ation, 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
coiled-coil 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
unli-kely 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
com-pletely ruled out, however
To provide evidence that it is the typical 3D
coiled-coil 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
stabil-ize the hydrophobic interface between helices forming
the coiled coil [31,32] These mutations strongly
influ-enced 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
deter-mines the self-association tendency Although residual self-association of DMPK ECMcould be demonstrated through covalent cross-linking, we conclude that this mutant provides a proficient tool to study multimeriza-tion-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 pep-tide substrate was two to threefold reduced when com-pared with the corresponding activities of wild-type DMPK E To us this suggests that DMPK auto-phosphorylation 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 intra-molecular autophosphorylation More detailed under-standing of the DMPK structure is needed to be able
to distinguish between these possibilities In DMPK-like 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 phosphoryla-tion may be a result of the experimental condiphosphoryla-tions 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 stabil-ize the DMPK ECM MYPT2 interaction with a chem-ical 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 inter-action 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
Trang 10C-terminal amino acids [24] Here, we show that
muta-tional 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
cau-ses increased exposure of YFP–DMPK C to the
proteo-lytic 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
two-step 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
poly-peptide 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
con-formation, 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
target-ing 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
hetero-multimerization of DMPK isoforms and that
multime-rization 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
trans-fected as described previously [24]
Expression plasmids and site-directed mutagenesis
Expression vectors for HA-, His- and EYFP-tagged DMPK A–F have been described previously [5,24] Expression plas-mids, encoding VSV-tagged DMPK truncation constructs, were obtained by cloning PCR fragments amplified from template pSGmDMPK E [15] with the use of Pfu poly-merase and specific primers EcoRI and XhoI sites were incorporated in the forward and reverse primers, respect-ively (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¢-ATAGAATTCATGTCAGCCGAAGTGCG-3¢ and 5¢-ATTCTCGAGTCAAGTGAGCCGGTCCTCCA-3¢; 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¢-ATACTCGAGCTAGGGATCTGCGGCT-3¢; 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 (Strata-gene, 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,
CCMand ECM, the following cloning steps were carried out First, an L515G mutation was introduced into pSGHADMPK A and C, as described above, resulting in pSGHADMPK A(L515G) and C(L515G) Then, two frag-ments [an AflII–BspEI fragment – common to all DMPK isoforms and including mutations L477G and L487G – iso-lated from pSGHADMPK ECM, and a BspEI–BsrGI frag-ment – 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, CCMand