Phosphorylation switches specific for the cardiac ... This phosphorylation switch can be mimicked .... isoforms while inter-species identity with the chicken fast.
The EMBO Journal vol.14 no.9 pp. 1952-1960, 1995
Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? Mathias Gautell, Orsetta Zuffardi2, Alexandra Freiburg and Siegfried Labeit European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany and 2Biologia Generale e Genetica Medica, Universita di Pavia, Pavia, Italy 'Corresponding author Communicated by Matti Saraste
Cardiac myosin binding protein-C (cardiac MyBP-C, cardiac C protein) belongs to a family of proteins implicated in both regulatory and structural functions of striated muscle. For the cardiac isoform, regulatory phosphorylation in vivo by cAMP-dependent protein kinase (PKA) upon adrenergic stimulation is linked to modulation of cardiac contraction. The sequence of human cardiac MyBP-C now reveals regulatory motifs specific for this isoform. Site-directed mutagenesis identifies a LAGGGRRIS loop in the N-terminal region of cardiac MyBP-C as the key substrate site for phosphorylation by both PKA and a calmodulindependent protein kinase associated with the native protein. Phosphorylation of two further sites by PKA is induced by phosphorylation of this isoform-specific site. This phosphorylation switch can be mimicked by aspartic acid instead of phosphoserine. Cardiac MyBP-C is therefore specifically equipped with sensors for adrenergic regulation of cardiac contraction, possibly implicating cardiac MyBP-C in cardiac disease. The gene coding for cardiac MyBP-C has been assigned to the chromosomal location lipll.2 in humans, and is therefore in a region of physical linkage to subsets of familial hypertrophic cardiomyopathy (FHC). This makes cardiac MyBP-C a candidate gene for chromosome 11-associated FHC. Key words: cardiac muscle/familial hypertrophic cardiomyopathy/myosin binding protein/protein phosphorylation/titin ligand
Introduction The contractile proteins of striated muscle sarcomeres, actin and myosin, are assembled into regular thin and thick filaments, respectively, which also contain several associated structural and regulatory proteins. The myosinassociated C and X proteins (later termed myosin binding protein-C and -X or MyBP-C; Vaughan et al., 1993) were discovered by Offer and co-workers (Offer et al., 1973) as co-purifying contaminants in myosin preparations. The MyBP-C family comprises isoforms specific for slow, fast and cardiac muscles (Yamamoto and Moos, 1983). Subsequent cloning of the skeletal isoforms of myosin binding protein (C and X protein) showed them to belong
to the intracellular immunoglobulin superfamily (Einheber and Fischman, 1990; Furst et al., 1992; Vaughan et al., 1992, 1993). The known skeletal isoforms all share a conserved domain pattern consisting of immunoglobulin-I set domains (Harpaz and Chothia, 1994; Ig-I or type II as by Benian et al., 1989) and fibronectin-IIl domains (fn-3 or type I) arranged in a pattern (Ig-I)-X-(Ig-I)4-(fn-3)2 -(Ig-I)(fn-3)-(Ig-I), X being a motif of function so far unknown (Furst et al., 1992; Vaughan et al., 1992, 1993; Weber et al., 1993). Immunoelectron microscopy techniques have revealed that MyBP in both slow and fast striated muscles are localized in a defined zone of the thick filament, the C-zone (Squire, 1981), where they are spaced regularly in single or double stripes at intervals of 43 nm (Craig and Offer, 1976; Dennis et al., 1984; Bennett et al., 1986). Analysis of developing myocytes has suggested that MyBP-C participates in the assembly of thick filaments in skeletal and cardiac muscles (Obinata et al., 1984; Schultheiss et al., 1990). Multiple protein-protein contacts are involved in this process: MyBP-C is intimately associated with myosin filaments. This interaction is directed by a C-terminal myosin binding Ig-I domain which is conserved within the whole family (Okagaki et al., 1993). Furthermore, MyBP-C binds to recombinant A-band fragments of the giant muscle protein titin (Labeit et al., 1992) as well as to native titin (Fuirst et al., 1992; Soteriou et al., 1993). Regulatory interactions with both F-actin and the motor domain of myosin, subfragment-1 (Moos et al., 1975, 1978; Starr and Offer, 1978; Moos and Feng, 1980), result in a modulation of myosin ATPase activity, while removal of C protein from skinned cardiac myofibrils was reported to result in increased active tension (Hofman et al., 1991). A potential regulatory role of C protein was also suggested by stimulation of metabolically 32P-labelled intact myocardium with adrenergic substances, which resulted in the incorporation of maximally 3 mol of phosphate per mol of cardiac MyBP-C and is paralleled by an increase in systolic tension development (Jeacocke and England, 1980; Garvey et al., 1988; Schlender and Bean, 1991). Both phosphorylation of cardiac MyBP-C and systolic tension are inversely affected by cholinergic agonists (Hartzell and Titus, 1982). Purification of the cardiac form of MyBP-C has been shown earlier to result in co-purification of a calmodulin-activated protein kinase activity (MyBP-C associated kinase), which was proposed to be Ca2+/calmodulin kinase II based on biochemical properties (Hartzell and Glass, 1984; Schlender and Bean, 1991). The physiological role of Ca2+/calmodulin-dependent phosphorylation of cardiac MyBP-C remains unclear to date. Nevertheless, cardiac MyBP-C appears not to have exclusively structural functions, but also senses regulatory signals possibly from several pathways linked through protein kinase cascades.
1 Oxford University Press 1 952
MyBP-C: a modulator of cardiac contraction?
We therefore obtained the primary structure of cardiac MyBP-C in order to identify the regions of cardiac MyBP-C phosphorylated by PKA during adrenergic regulation. This provides a basis for the detailed investigation of its interactions with the contractile proteins of the sarcomere and their regulation .
A
M [kDa] M 1 2 3 4
M lkDa] 1
My c
2 3 *41' e 1
My
Results Purification and molecular cloning of cardiac MyBP-C MyBP-C from rabbit heart was purified (Figure 1A) as described in Materials and methods. Attempts to determine the N-terminal sequence of the purified protein failed, apparently due to N-terminal blockage. A polyclonal rat serum (R 1) was raised against the protein which recognized a single band in Western blots of rabbit cardiac muscle (Figure 1B) and stained sarcomeric A-bands of rabbit heart in immunofluorescence (not shown). Aliquots of RI were used for screening a human heart cDNA expression library and seven independent clones could be isolated. The two clones with the largest inserts were both sequenced, found to overlap and together to cover 3.3 kb of cDNA. The sequence indicated that the cDNA was incomplete at the 5' end, necessitating 5' cDNA extensions. After two rounds of 5' extension and sequencing, all four partial clones could be assembled into a 4.5 kb contig which appears to correspond to the full length cardiac MyBP-C cDNA. At its 3' end, a 587 bp untranslated region contains a polyadenylation signal and a poly(dA) tail. At the 5' end, a start ATG is in-frame with the single large open reading frame (ORF) and is preceded by stop codons in all three reading frames. To show that the sequence corresponds to the cardiac MyBP-C, two internal proteolytic subfragments from the purified cardiac MyBP-C were N-terminally sequenced. The peptide sequences match the sequence predicted from the cDNA (underlined in Figure 2). Furthermore, a polyclonal antibody (I1) was raised against the expressed N-terminal Cl -C2 fragment (see below). Immunoblot detection with I1 recognizes the same band in Western blots (Figure 1B) and shows the same staining pattern in immunofluorescence (not shown) as the RI serum used for expression cloning. Also, I1 detects no cross-reactive protein in adult tissues, other than in heart muscle (not shown). We conclude that the cloned cDNA sequence and the encoded protein are cardiac specific and correspond to cardiac MyBP-C. The full-length 4.5 kb cDNA of cardiac MyBP-C contains a single large ORF encoding a 1173 residue polypeptide. The deduced amino acid sequence was examined for similarity to the known MyBP. As in the skeletal isoforms, seven Ig-I modules and three fn-3 modules could be identified in the primary structure. In addition, a 103 residue motif between the second and third Ig-I domain was present that was specific only to MyBP-C. The Ig-I and fn-3 were successively numbered Cl -C 10, in analogy to the nomenclature introduced by Vaughan et al. (1993). The MyBP-C-specific sequence will be referred to here as the MyBP-C motif. Overall sequence identity is as high as 55% for the human skeletal isoforms while inter-species identity with the chicken fast skeletal sequence (Einheber and Fischman, 1990; Weber
B
C
A-tin f-%t,Lli 14w
97 .
67
ft
am
43
41VP W1
31
-
20
-
L
14 --
Fig. 1. (A) SDS-polyacrylamide gel electrophoresis on a 4-18% gradient gel of fractions from the purification of rabbit cardiac MyBP-C. Lane 1: total rabbit heart; lane 2: high-salt myofibrillar extract after dialysis to buffer C; lane 3: MyBP-C fraction from BioGel column; lane 4: cardiac MyBP-C after ion-exchange chromatography on monoQ. M: marker proteins (97 kDa: phosphorylase; 67 kDa: bovine serum albumin; 43 kDa: ovalbumin; 31 kDa: carbonic anhydrase; 20 kDa: soybean trypsin inhibitor; 14 kDa: lysozyme). My: myosin (215 kDa), C: C protein. (B) Western blot analysis with the anti-cardiac MyBP-C sera RI and the serum against the recombinant N-terminus I1. Lane 1: total rabbit heart blot stained with amido black; lane 2: as 1, probed with RI serum; lane 3: as 1, probed with Il serum.
et al., 1993) reaches 58%. This conservation shows two exceptions: cardiac-specific additional sequences are found in the MyBP-C motif and in the Ig-I module C5 (Figure 2). Furthermore, the proline-alanine-rich N-terminal peptide is highly variant within the entire family of MyBP-C.
Phosphorylation of cardiac MyBP-C is localized in the N-terminal region To identify the regulatory elements of cardiac MyBP-C, phosphorylation assays of purified rabbit cardiac MyBP-C (monoQ fractions) were performed essentially as described by Schlender and Bean (1990). The protein from rabbit heart could be stoichiometrically phosphorylated at a ratio of 1.2 + 0.2 mol of phosphate/molecule (based on the calculated molecular weight of 137.2 kDa) by the copurifying kinase activity. This phosphorylation is strictly calmodulin dependent and can be inhibited by EGTA or the calmodulin binding peptide M13 (Blumenthal et al., 1988). The MyBP-C-associated kinase activity is lost after gel filtration, as described in Materials and methods. Addition of the catalytic subunit of cyclic AMP-dependent kinase leads to maximal phosphate incorporation of 3.2 + 0.15 mol/mol protein. These results for the mammalian protein are in good agreement with those obtained previously for avian MyBP-C (Schlender and Bean, 1991). In order to gain information about the regions of cardiac MyBP-C that are phosphorylated by PKA, proteolytic fragments of the radiolabelled protein were generated with 1953
M.Gautel et al.
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
proline-rich region * 1 MPEPSKAAPKKEAKKKEEKKEEKKEAPPPQEH.....KDEAPDDVHPPETPDP
Cl IgI
MPEAKPAAKKAPKGKDAPKGAPKEAPPKEAPAE.A....PKE.APPEDQSPTAEEP
FL
ILFI KPQGG
MPEPTKKEENEVPAPAPPPEEPSKEKEAGTTPAKDWTLVETPPGEEQAKQNANSQ MLAPAPAPAEATGAPGEAPAPAAELGESAPSPKGS .... SSAALNGPTPGAPDDP
IKWFKGKW4 I R S K FKGKWI
PQDG.
L
* MyBP-C motif
101 LGD
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
100 PQKW PDSV. AWKK
LGS LAS DLSSK v2LQ
.......
*C2lgl
201
Chk_F-Mybpc
Li&'PLJJAY 'r "-L QVF ilVRFrELALvK t'IKr-O TVEI _YDUThU[REA
MAK EYEKIAFQYGITDLRGMLKRL LLKKR EVEEEKKKKKKDDDDLGIPP WELL WELL .AK EYEKIAFQYGITDLRGMLKRL LLKRR EKQQEEEPQV .......... LLKKRIjSFRTPRDSKLEAPAE.... EVEILRAPP EYERIAFQYGVTDLRGMLKR
K
LDPAY
KK
T r%nA %?
v
SD D
,,
,
* C3 IgI
301
400
---. _-2: L1 ELWYKNG 4LRR . CYVFE VKILnTIK . ILT YVF L KNG 1 YIF 414 ILF O4 L r1KWYKNG 4*. AE S.+YIF -
.KWLKNGd+JL
A
*
401 Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
300
RD .LPPEAoWI AVTKKI;EYERIAFOYGITDLRGMLKRLKIK 9ZDYnK-AEO t-rT.T. Evurvv KKKKKD EDDOFF.
F-Mybpc S-Mybpc C-Mybpc Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
200 G DF GLDF GLDF GDF
KSPR..SSEGNVL CS PRQ.DASGQSLEIFKRTSI.....EKK F S ESTGTTPNIDIR AFKRSG4......EG F C EAMG.TGDLDLL R L IDSH
.DDKFET KVAMGD VLH KVLGD KE SA KE RHS44IFEMQII KAKDNF HDS YDRASKmJIFELHI TDAQPAF
RH
C4 lgl
RDDAFK..VKF414a4H fTEAELSP F I DVVQ RWF LTREDSFKARYRFP
500 s
ASEVAEP
PSKWKN PS 1 ASEQAEQ KCEVS 1.LhIA4AEW ES GKEICKCEIS Qf II|PGP..KSR RI FI GATK EAMLDQKCEVSPD LTREETFK..YRFPR j * linker *C5 lgl 600 501
EGFLDLCKVEY
ISG L P PKQ1PK P EEGDF DG'LG * LNFL1 KVEYVPKQ PKAIVVVAGNKLRLITGE. IAGNKLRLEISGEP DA. LT EGD VHVI.......... PK UI IVVVAGNKLRL ISGD .C'KIDFVPRQI RVHKB2LT1TP EAD EMK -C
RFHKLV Chk_F-Mybpc |I-. H>RFHKLV F-Mybpc RIHKLV S-Mybpc C-Mybpc 601 Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
A GDQL@ATEEPEW A L _ GDETRF kR iNIEEFGD
. .. .. .. . . .... .. . . . . . .. .. . . ... .. .. .. . ... . . ... .......................... GD
* C>6fn3
V
701 WI MWEPEI YL
VNF
YLMERK VERKKK w
IYFIERK
WEPPIp
t I
T
W14( F
NWSt FD
801 KWRPPERIGAGGVDG
KWRPPNRIGAGGIDGEVEYY. WRPPDHIGAAGLDG YLEY
TT
EMRVFAVNAIGV
FMPI
ISEIL
TT
MI EGVEVRIFAVNAIGI
FVPL
SPIT
P
KWRPPERVGAGGLDGVEY
ILFR FVR
G
TDT r TDT
PRHLRQTY
GRT.TL YY
PKI
PVT PI
GA
TVT
PKI
PRHLRQTY PRHLKQTY
R
PRHLRQTI
*C9 fn3
901 PAF )TS
VIPFQGKPRPQV
Chk_F-Mybpc F-Mybpc
NLVPFQGKPRPQV 1001
AT |
FVR
IFIR
S
E ID
S
G I VKI
C10 IgI
FTIN
r
*
1100
1101
R
r,
pLS4tAT!T~A 11V ;FSI1AA~T
SMEWF
tTIVOATMEWF
S
1000
fl 7fWE1 ikRFR -qDhA fl [rARFT RR .qfl '~-vk -IRFA1 -Fl'T-'ILi RVYTEN tLS;PG r RLIV TMEWFtE F5Y VQ GT PF E pHFL L
IDRiI I I 17W21 dl fl-Fk1 I I I IT. flchl Jv m ~DUvyP?qI
A
V
V V
v
S
C)KN VIPFQGKPRPEL GE LIPFQGKPRPQGE
S-Mybpc C -Mybpc
'r
* C8 191 goo
LLFRT
L DKTtr IT QGLEVK
WI
V
r1FMPI
EMRVYAVNAIGM
M
TEL AR JTEP RC4IrVL
E
C7 fn3 800 ET LTDT EMRVFAVNAG;FPI
MI MIEGI
L
TDI
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
PDFPG
S I6D
I liFDPD PuHGs Ay A|^YE$LVEDJI+A VPGT TQGNKAPARPAPDAPZNGD3WDKK@ET
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
Chk_F-Mybpc F-Mybpc S-Mybpc C-Mybpc
700
G
R
INtDFAtT
IU1TXtPKAPtArIFTk'
1189 TTFCRLVRV Q*
Fig. 2. Multiple sequence alignment of human cardiac MyBP-C with the skeletal isoforms from human and chicken. The first amino acid of each module is marked by an asterisk above the sequence. The consensus of the tryptic peptide sequences are underlined. The domains are consecutively numbered C -CO0 with their subclasses marked. Additional cardiac-specific sequences are highlighted in bold letters. Chk_F-Mybpc: chicken fast skeletal MyBP-C (Weber et al., 1993); F-Mybpc: human fast skeletal MyBP-C (Fuirst et al., 1992); S-Mybpc: human slow skeletal MyBP-C (Fuirst et al., 1992); C-Mybpc: human cardiac MyBP-C.
chymotrypsin at an enzyme/substrate ratio of 1:800 at 37°C for 20 min at pH 8 and were investigated for phosphorylation by autoradiography. Labelled fragments of apparent molecular weights of 120, 70, 58 and 37 kDa were detected when rabbit cardiac MyBP-C was phos1954
phorylated in the presence of [y-32P]ATP and subsequently subjected to limited proteolysis. The 70 and 37 kDa fragments were analysed by microsequencing. Both fragments were found to be N-terminally blocked. Immunoprecipitation of labelled cardiac MyBP-C fragments
MyBP-C: a modulator of cardiac contraction?
Al MlkDa]
M
2
1
97 67
3
MyBP-C and calmodulin, phosphate incorporation into serine residues at a stoichiometry of 1.1 ± 0.1 mol/mol of protein was observed (not shown). We conclude from these experiments that incorporation of maximally three phosphate residues into cardiac MyBP-C by PKA or one phosphate residue by MyBP-C-associated kinase occurs within the N-terminal Cl -C2 fragment.
5
4
_. _
43 31
20 14
fM [kDa] M
1
2
3
4
5 6
7
8
9 10 11 12
97 67
43
31
-
20 14
Fig. 3. (A) Phosphorylation of proteolytic fragments of rabbit cardiac MyBP-C phosphorylated by PKA. Lane 1: Coomassie-stained gel of MyBP-C before proteolysis; lane 2: limited chymotryptic digest of MyBP-C; lane 3: autoradiograph of duplicate of lane 2; lane 4: autoradiograph of PKA-labelled fragments immunoprecipitated by the Il serum; lane 5: autoradiograph of MyBP-C-associated kinaselabelled fragments immunoprecipitated by the Il serum. (B) Phosphorylation of overlapping recombinant fragments of human cardiac MyBP-C by PKA and MyBP-C-associated CaM-kinase. Lanes 1-4: Coomassie stain of fragments Cl-C2, C2-C5, C5-C8, C8-CIO. Lanes 5-8: autoradiograph of the corresponding lanes, phosphorylated by PKA. Lanes 9-12: corresponding lanes as 1-4, phosphorylated by MyBP-C-associated kinase.
(Materials and methods) with I1 serum raised against the recombinant N-terminal Cl -C2 fragment leads to precipitation of all labelled fragments (Figure 3A). These results indicate that phosphorylation of native MyBP-C is localized in the N-terminal region. To obtain a more characterized map of phosphorylation, four overlapping fragments representing all modules of cardiac MyBP-C were expressed in Escherichia coli (C1-C2, C2-C5, C5-C7, C7-C10) and tested for phosphorylation by PKA and MyBP-C-associated kinase. No significant label incorporation was observed in any construct beyond Cl -C2 (Figure 3B). Mass spectroscopical analysis of C1- C2 phosphorylated preparatively by cardiac PKA at 0.1 U/jg and excess ATP shows di- and triphosphorylated protein, which confirms the stoichiometry of three sites obtained for the native protein (Figure 4). When Cl -C2 was incubated with native
Three hierarchically accessible phosphorylation sites in the MyBP-C motif The sequence of Cl-C2 predicts four potential phosphorylation sites for PKA (labelled A-D in Figure 5A) based on the criteria compiled by Pearson and Kemp (1991), all of which are localized in the MyBP-C motif (residues 157-259). To identify which of the predicted sites are actually substrates for PKA, site-directed mutants were constructed that replaced the predicted phosphorylation residues with alanine (ASA, BSA, CSA, DTA). When these constructs were phosphorylated by cardiac PKA, a 30-40% decrease in phosphorylation intensity was observed for the mutants of sites A and C. Mutation of site D, with a putative threonine-directed phosphorylation, resulted in no significant reduction of phosphate incorporation (Figure SB). This site is presumably well packed or shielded in the adjacent domain and hence too inaccessible. Furthermore, phosphoamino acid analysis of C 1-C2 phosphorylated by PKA demonstrates phosphate incorporation into serine residues only (Figure SC). Mutation of site B in the cardiac-specific LAGGGRRIS insertion, however, resulted in a marked decrease of phosphate incorporation by PKA to -9% (Figure SB). To test the hypothesis that the LAGGGRRIS insertion may be a cardiac-specific conformational switch which makes the two other mutationally defined sites accessible when it is phosphorylated, two further mutants were constructed. In the deletion mutant Cl - C2AL, this motif was removed to result in a 'skeletal'-like domain. In the mutant Cl -C2/BsD, the serine phosphorylation site was replaced by aspartic acid to potentially mimic the negative charge of phosphoserine. Phosphorylation assays with Cl -C2IL in comparison to the wild-type resulted in a relative phosphate incorporation of 8% for the mutant by PKA. The mutant Cl -C2/BSD, however, showed significantly higher levels of PKA phosphorylation (55%) in maximally two sites, and is therefore constitutively activated (Figure 5D). However, all mutants in site B show phosphate incorporation below 10% by the MyBPC-associated calmodulin-activated kinase (not shown), implicating site B as the main target site for calmodulindependent phosphorylation.
Chromosomal localization of cardiac MyBP-C The chromosomal localization of the gene for cardiac MyBP-C was determined by fluorescent in situ hybridization (FISH) as described in Materials and methods. In the 83 metaphases analysed, 240 signals were associated with chromosomes: 118 (49%) were located at l1pll.2, and the distribution of the remaining 122 signals was random (Figure 6). This suggests that, under the hybridization conditions used, no other members of the MyBP-C gene family cross-hybridize. These data were confirmed by a second FISH experiment in which the same probes were hybridized to the metaphases of a patient with a balanced 1955
M.Gautel et al.
io
36,200
36,250
Molecular Weight (Da) Fig. 4. Mass-spectroscopy of C1-C2 phosphorylated by PKA. The observed mass of the unphosphorylated Cl -C2 construct is 34 819 Da; after phosphorylation by PKA, only peaks corresponding to the di-(34 979.5 Da) and triphosphorylated (35 060.2 Da) forms are detectable apart from the ion adducts indicated.
translocation t(1 1;20) (p1 1.3;q 11). In all the 20 metaphases suitable for FISH analysis, hybridization signals were unequivocally localized at the proximal region of the short arm of chromosome 11 and of the der( 11) (data not shown). The physical mapping of cardiac MyBP-C to ilpl l.2 co-localizes the gene to a region of physical linkage to cases of familial hypertrophic cardiomyopathy (FHC; Carrier et al., 1993).
Discussion We present here the complete primary structure of the cardiac isoform of myosin binding protein C and the identification of its regulatory phosphorylation sites. The complete cDNA of cardiac MyBP-C encodes a 1173 residue polypeptide, with a predicted molecular weight of 137 kDa. This is smaller than the apparent molecular weight in SDS-PAGE; abnormal electrophoretic motility has also, however, been described for a number of closely related proteins (Vaughan et al., 1993; Vinkemeyer et al., 1993), and might be caused by the unusual prolinealanine-rich N-terminal sequence. Cardiac MyBP-C shares the overall modular architecture of the skeletal myosin binding proteins (Figure 2). However, two distinct regions discriminate the cardiac isoform and implicate its involvement in regulatory functions of cardiac contraction. The first domain of the intracellular immunoglobulin-I set (Harpaz and Chothia, 1994) is linked to the next Ig-I module by a 103 residue stretch (residues 157-259) with no homology to other protein families and which we called the MyBP-C motif. It contains no pattern that allows it to be classified as one of the constituent modules of MyBP-Cs and is therefore likely to adopt a distinct
1956
fold. The MyBP-C motif displays, however, a remarkable degree of inter-species and inter-isoform conservation, signifying a crucial functional role (Figure 2). The alignment of skeletal and cardiac MyBP-C sequences reveals the addition of a nine residue long loop in the cardiac MyBP-C motif with the sequence LAGGGRRIS. This RRIS motif, preceded by a probably highly flexible sequence, is a potential phosphorylation site for a number of protein kinases (Pearson and Kemp, 1991). We could demonstrate that phosphorylation of cardiac MyBP-C by cardiac PKA and MyBP-C-associated kinase is restricted to the N-terminal region (Figure 3) and to the region between C l and C2. Four putative phosphorylation sites are predicted in the MyBP-C motif in Cl -C2 (Figure 5A), three of which are phosphorylated by PKA as shown by mutational analysis and phosphoamino acid mapping (Figure SB and C). Interestingly, mutation of site B in the LAGGGRRIS insertion results in functional inactivation of the two other sites as well. This argues that, upon phosphorylation of site B, conformational changes are induced that make two further sites accessible. This is supported by the finding that mimicking the phosphoserine by introduction of an acidic amino acid in place of serine in the mutant Cl -C2/ BSD can restore the accessibility of the two further phosphorylation sites, most probably by inducing a conformational change in the flexible loop insertion (Figure SD). This strategy has already been employed successfully to mimic activating phosphorylation in the MAP kinase kinase activation loop (Pages et al., 1994). To gain further evidence that the LAGGGRRIS loop controls access to the neighbouring sites A and C, the deletion mutant C l-C2AL, in which this loop was specifically removed, was modelled on the basis of
MyBP-C: a modulator of cardiac contraction?
sequence homology to the skeletal forms of MyBP-C. This was expected to result in an essentially 'nonregulated' domain for Cl- C2AL. Indeed, phosphorylation by both PKA and MyBP-C-associated kinase drops to the levels of the serine-alanine exchange mutant (Figure 5D).
A loop insertonr,
_e r --_' RfzrPSEYSR AF. Ys.'LRSML'5K
,
.D.
B
120c 100r.
80
_
a
L
60 40
-
HH0
20 -
C
B
A
D
WT sites
Cr
P-S
P-T
P-Y
D 5 1 04 4 104
- AL-aSA ~---- SD
-
3 10
-
2 10 -
.-
-
4W
.
1 1 04 -
.
1t
15
z
:- T-:
20
25
T
30
fl
35 min
We therefore conclude that the cardiac-specific loop LAGGGRRIS represents the major regulatory phosphorylation site of MyBP-C for PKA and the associated calmodulin-activated protein kinase. This also identifies the MyBP-C motif as the regulatory module of the protein family. Phosphate turnover on cardiac MyBP-C in intact heart is highly dynamic and linked to changes in contraction properties (Jeacocke and England, 1980; Hartzell and Titus, 1982; Garvey et al., 1988). We therefore propose that the MyBP-C motif is involved in mechanisms where phosphorylation of MyBP-C in cardiac muscle in vivo alters muscle function, such as contraction-relaxation rates, rather than structural functions, and is specialized for the dynamic regulation of cardiac contractility by the isoform-specific phosphorylation site in the LAGGGRRIS loop. Interestingly, only the human fast skeletal isoform shows sequence homology to the phosphorylation site A of the cardiac isoform (Figure 2). Whether this site is involved in the phosphorylation of skeletal MyBP-C, as has been suggested by biochemical investigations (Starr and Offer, 1982), remains to be investigated. The second distinct sequence addition specific for the cardiac isoform is localized in the Ig-I domain C5. Here, based on the homology to the structure of telokin (Holden et al., 1992), the predicted loop connecting 13-strands C and D contains a 28 residue stretch abundant in proline and charged residues (Figure 2). An insertion of such size in this hypervariable loop has not been described in intracellular Ig-I modules so far, and this finding once more underlines the great amount of functional plasticity that the V-frame fold of immunoglobulin domains allows as a stable scaffold for binding motifs (Williams and Barclay, 1988; Williams et al., 1989). Proline/charge-rich sequence stretches are increasingly identified as the basis of specific ligand interactions of signal transduction molecules, e.g. the ligands of SH3 domains (Musacchio et al., 1994, and references therein), and it will therefore be of interest to identify the binding partner and structure of this specialized domain. As this loop insertion is strictly cardiac specific, we propose that it provides a binding scaffold for other cardiac-specific ligands of MyBP-C, possibly binding the co-purifying kinase activity, the identity and functional role of which remains to be investigated. The regulatory and structural functions of cardiac MyBP-C raise the question of its involvement in cardiac disease. The genomic localization of human cardiac MyBP-C has been assigned to lip11.2 and is therefore physically linked to genetic markers for the chromosome 11-associated form of FHC (Carrier et al., 1993). Therefore, and together with its cardiac-specific expression, the cardiac MyBP-C gene is a candidate gene for FHC. The previous identification of ,-myosin in the chromosome Fig. 5. (A) The cardiac MyBP-C regulatory domain. The putative phosphorylation sites A-D are underlined and the mutationally defined sites are highlighted in bold letters. The LAGGGRRIS loop is marked by a line. (B) Quantitative analysis of PKA phosphorylation of Ser-4Ala and Thr-*Ala mutants in sites A -D. WT: wild-type Cl -C2. Means of two experiments are shown. (C) Phosphoamino acid analysis by TLC of Cl -C2 phosphorylated by PKA. The positions of the marker amino acids are shown. (D) Time-course of PKA phosphorylation of equal concentrations of mutants of Cl -C2 in the LAGGGRRIS loop. C1 -C2/BSD (SD), Cl -C2SA (SA), Cl -C2AL (AL) and the wild-type construct (WT).
1957
M.Gautel et al.
000000 AkAKA.AmhAmk---. weewe-,
-
Fig. 6. Left: (a and c) partial metaphases with hybridization signals at lp1 1.2 (arrows). (b and d) DAPI banding of the same metaphases. Right: distribution of hybridization signals on chromosome 11. *: 10 signals; *: I signal.
14q1-linked form of FHC (Jarcho et al., 1989) also involves one of the two known ligands of cardiac MyBP-C. This would further confirm the concept proposed by Thierfelder and co-workers that FHC is a disease of the sarcomere (Thierfelder et al., 1994). The availability of the complete sequence of cardiac MyBP-C will now allow us to investigate whether cardiac MyBP-C is indeed pathogenetically involved in chromosome 1 I-associated FHC. The recombinant regulatory domain of cardiac MyBP-C and its mutants will also allow interactions with the contractile machinery to be mapped, and the regulatory effects of MyBP-C phosphorylation to be studied in detail.
MyBP-C eluted at -220 mM salt. On gel filtration on Superose 6 (Pharmacia) in 300 mM KCI, 20 mM sodium phosphate, 1 mM EDTA, 1 mM DTT pH 7, cardiac MyBP-C eluted at the retention volume consistent with a monomeric protein of -140 kDa. These fractions no
Materials and methods
The anti-cardiac MyBP-C antibody was diluted 1: 1000 and used to screen a human heart cDNA expression library (Stratagene #936208) essentially as described (Nierendorf et al., 1987). Positive clones were subjected to two rounds of re-screening and plaque purified. Inserts of purified clones were subcloned into M13 (Yanish-Perron et al., 1985) and sequenced (Sanger et al., 1977). The immunopositive cDNAs were found not to cover the entire message. For extension into both the 5' and 3' direction, anchored PCR methods were used (Saiki et al., 1985; Rasmussen et al., 1989). The cDNA sequence has been deposited in the EMBL data bank, accession number X84075.
longer show the associated kinase activity.
Immunological methods Fractions from monoQ columns were concentrated by ultrafiltration and subjected to SDS-PAGE. The 165 kDa region was electroeluted using an electroelution device from BioRad. Inbred female Wistar rats were immunized using a standardized scheme. Standard immunological methods were used for blot detection. Indirect immunofluorescence was performed as described (Fuirst et al., 1988) with serum dilutions of 1: 100 using a goat anti-rat fluorescein isothiocyanate (FITC) conjugate (Sigma).
cDNA cloning and sequencing
Purification of cardiac MyBP-C Purification of cardiac MyBP-C from rabbit left ventricles was performed by a modification of the methods for MyBP-C described by Hartzell and Glass (1984) and Fulrst et al. (1992). Briefly, rabbit hearts were excised immediately post-mortem and left ventricles were dissected on ice. For MyBP-C extraction, 10 g of tissue were homogenized in icecold extraction buffer (100 mM KCI, 20 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol (DTT) and a cocktail of protease inhibitors as described by Furst et al. (1992), and myofibrils were sedimented at 3000 g. The addition of protease inhibitors in all steps is necessitated by the rapid proteolysis of cardiac MyBP-C by endogenous proteases. The fibril pellet was washed four times in 10 volumes of the above buffer and subsequently extracted in high-salt phosphate buffer A (50 mM sodium phosphate pH 6.5, 2 mM Na4P207, 300 mM KCI, 1 mM EDTA, 1 mM DTT and protease inhibitors) for 4 h on ice. The extracted myofibrils were sedimented at 10 000 g and the supematant dialysed against buffer B (as A without KCI) to precipitate extracted myosin. The supernatant after dialysis was concentrated by ammonium sulphate precipitation at 45% saturation. The pellet was dissolved in buffer C (10 mM sodium phosphate pH 7, 0.3 M KC1, 0.1 mM EDTA and protease inhibitors) and fractionated on a hydroxylapatite column essentially as described (Hartzell and Glass, 1984). C protein-containing fractions were pooled and further fractionated on a monoQ column (Pharmacia) as described (Fulrst et al., 1992). Cardiac
1958
Sequence analysis and interpretation Sequence editing, fragment assembly, prediction of ORFs and dot-plot self-comparison were performed with the UWGCG software package (Devereux et al., 1984). The single predicted ORF in the human cardiac MyBP-C contig was used to translate the encoded peptide, and screened for Ig-I and fn-3 modules by eye. Segments not consisting of modular sequences were used to search the Swiss Prot data library (release 19) with FASTA. Motifs were aligned by GCG-PILEUP and CLUSTAL-W (Thompson et al., 1994) and edited by eye, and conserved positions were boxed with PRETTYPLOT (P.Rice, EMBL).
Expression and mutagenesis of cardiac MyBP-C sequences in E.coli The overlapping fragments of MyBP-C spanning all Ig-I and fn-3 domains and the MyBP-C motif were isolated by PCR (Saiki et al.,
MyBP-C: a modulator of cardiac contraction? 1985) using the original kZAP phage isolates as templates. Domain boundaries for expression constructs were chosen based on the stability criteria established for titin type II modules (Politou et al., 1994a,b). The fragments obtained were subcloned into the pET8c vector (Studier et al., 1990) and fused N-terminally with an oligonucleotide linker encoding a His6 tag sequence. Mutants were constructed by PCR assembly of the mutagenized N-terminal cassette with C2 and verified for correct folding by mass spectroscopy and DNA sequencing. After induction of transformed BL21[DE3]pLysS cells (Studier et al., 1990) with 0.1 mM IPTG for 4 h at 37°C, the harvested cell pellet was sonicated in 50 mM sodium phosphate pH 8.0, 500 mM NaCl, 0.2% Triton X-100 and a cocktail of protease inhibitors. After centrifugation at 25 000 g, soluble expressed products from the supernatant were purified by metal chelate affinity chromatography on Ni2+ NTA agarose (Qiagen) essentially as described (LeGrice and Gruninger-Leitch, 1990) and further purified by anion exchange chromatography on a monoQ column (Pharmacia). Folding of domains was verified by circular dichroism (CD) spectra in the far ultraviolet on a Jasco J-710 spectropolarimeter as described
(Politou
et
al., 1994a).
Phosphorylation assays PKA assays were carried out with 0.01 U PKA from porcine heart (Sigma) per ,ug protein in 20 [.l kinase assay buffer (25 mM HEPES pH 7.2, 100 mM KCI, 10 mM MgCl2, 0.1 mM CaCI2, I mM DTT, 0.2 mM ATP and I ,uCi [y-32P]ATP, 3000Ci/mM). For phosphoamino acid analysis, 0.1 U PKAltg were used to label all sites quantitatively. Phosphorylation assays with MyBP-C-associated kinase were carried out using a final concentration of 0.1 mg/ml of the substrate proteins, native C protein (0.1 ,ug) and 20 nM bovine brain calmodulin (Sigma). Assays were started by addition of a lOx ATP mix, incubated at 30°C for 15 min, stopped by addition of 10 ,ul of sample buffer (Laemmli, 1970) and analysed on 15% polyacrylamide gels as described (Laemmli, 1970). Gels were autoradiographed at -80°C with intensifying screens for 12 h. Quantitation was carried out after precipitation on nitrocellulose filters with 5% trichloroacetic acid (TCA) by liquid scintillation in Ready Safe (Beckman) in a Beckman LS 8100 liquid scintillation counter against background and radionucleotide references for calibration. Means were calculated from two independent experiments. The concentration of the M13 peptide (a gift by G.Trave, EMBL) was 100 nM. Immunoprecipitations with the rat serum II were carried out using rabbit anti-rat IgG (Sigma) absorbed to protein-A beads (Pharmacia) at 0.25 jig/jil gel and blocked with 1% bovine serum albumin. Phosphorylation assays containing 2 jig cardiac MyBP-C were incubated with 5 jl of II serum and 15 jl anti-rat IgG beads for 30 min on ice, washed extensively with assay buffer and eluted with 20 jil SDS sample buffer. Phosphoamino acid analysis of radiolabelled protein was carried out essentially as described by Boyle et al. (1991). 10 jig of Cl -C2 from phosphorylation assays as above was precipitated by TCA and hydrolysed at 1 10C in 5.7 M HCI for 2 h. The hydrolysate was lyophylized and aliquots applied on Kodak TLC cellulose sheets. Phosphoamino acids were separated in one dimension by electrophoresis in a pyridine/acetic acid/water system (4:40:756) pH 3.5, at 750 V for 40 min. Standard phosphoamino acids (Sigma) were visualized by ninhydrin and marked. Labelled phosphoamino acids were then visualized by exposure to Kodak X-AR 5 film, marked and identified by comparison to the standards.
Chromosomal localization of cardiac MyBP-C FISH was performed with a pool of four PCR fragments of MyBP-C spanning the entire cDNA to metaphases of two normal males and of a male with a balanced translocation (t ll;20) (p ll.3;ql 1). The fragments (200 ng/slide) were labelled by nick translation (Boehringer Mannheim) with biotin- I 6-dUTP according to the supplier's protocol. Hybridization was performed as described (Rossi et al., 1993) at 37°C in 50% formamide/2X SSC with 5OX salmon sperm DNA and was followed by post-hybridization washes at 37°C in 50% formamide/2X SSC (3x5 min) and then in 2x SSC (15 min). Detection was performed by incubation of the slides with FITC-avidin (ONCOR detection kit) according to the supplier's instructions with two amplification steps. Chromosomes were counterstained with propidium iodide (I gg/ml), banded with diamidinophenyleneindole (DAPI) (Schweitzer, 1981) and subsequently mounted in anti-fading solution (Vectashield mounting
medium, VECTOR). Hybridization analysis was performed on metaphases with no more than 10 signals. A third amplification step was then done as described (Rossi et al., 1993) to obtain photographs with brighter signals although, in these conditions, the level of background was much higher.
Acknowledgements We are greatly indebted to Mathias Wilm for superb mass spectroscopy services. We would also like to thank Tony Houthaeve for N-terminal sequencing of MyBP-C fragments and Viviane Adam for oligo synthesis services. We are particularly grateful to David Stoddard for expert animal immunization and to Serge Roche for assistance with phosphoamino acid analysis. Without the generous support of Annalisa Pastore and Matti Saraste, this work would not have been possible. We are grateful to B.Bullard, T.Gibson, A.Pastore and A.Politou for fruitful discussions and critical reading of this manuscript. This work was supported by the grants from the Deutsche Forschungsgemeinschaft (MG, Ga-405/2-1; SL, La-668/2-2) and the EU.
References Benian,G.M., Kiff,J.E., Neckelmann,N., Moerman,D.G. and Waterston,R.H. (1989) Sequence of an unusually large protein implicated in the regulation of myosin activity in Celegans. Nature, 342, 45-50. Bennett,P., Craig,R., Starr,R. and Offer,G. (1986) The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle. J. Muscle Res. Cell Mot., 7, 550-567. Blumenthal,D.K., Charbonneau,H., Edelman,A.M., Hinds,T.R., Rosenberg,G.B., Storm,D.R., Vincenzi,F.F., Beavo,J.A. and Krebs,E.G. (1988) Synthetic peptides based on the calmodulin-binding domain of myosin light chain kinase inhibit activation of other calmodulindependent enzymes. Biochim. Biophys. Res. Commun., 156, 860-865. Boyle,W.J., van der Geer,P. and Hunter,T. (1991) Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol., 201, 110-149. Carrier,L., Hengstenberg,C., Beckmann,J.S., Guicheny,P., Dufour,C., Dausse,E., Berebbi-Bertrand,I., Wisnewsky,C., Bercovici,J., Pulvenis,D., Fetler,L., Vignal,A., Weissenbach,J., Hillaire,D., Feingold,J., Bouhour,J.-B., Hagege,A., Desnos,M., Isnard,R., Duborg,O., Komajda,M. and Schwartz,K. (1993) Mapping of a novel gene for familial hypertrophic cardiomyopathy to chromosome 11. Nature Genet., 4, 311-313. Craig,R. and Offer,G. (1976) The location of C-protein in rabbit skeletal muscle. Proc. R. Soc. Ser B, 192, 325-332. Dennis,J.E., Schimizu,T., Reinach,F.C. and Fischman,D.A. (1984) Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies. J. Cell Biol., 98,1514-1522. Devereux,J., Haeberli,P. and Smithies,O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res., 12, 387-395. Einheber,S. and Fischman,D. (1990) Isolation and characterization of a cDNA clone encoding avian skeletal muscle C-protein: an intracellular member of the immunoglobulin superfamily. Proc. Natl Acad. Sci. USA, 87, 2157- 2161. Fiirst,D.O., Osborn,M., Nave,R. and Weber,K. (1988) The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line. J. Cell Biol., 106, 1563-1572. Furst,D.O., Vinkemeyer,U. and Weber,K. (1992) Mammalian skeletal muscle C-protein: purification from bovine muscle, binding to titin and the characterization of a full-length cDNA. J. Cell Sci., 102, 769-778. Garvey,J.L, Kranias,E.G. and Solaro,R.J. (1988) Phosphorylation of Cprotein, troponin I and phospholamban in isolated rabbit hearts. Biochem. J., 249, 709-714. Harpaz,Y. and Chothia,C. (1994) Many of the immunoglobulin superfamily domains in cell adhesion molecules and surface receptors belong to a new structural set which is close to that containing variable domains. J. Mol. Biol., 238, 528-539. Hartzell,H.C. and Glass,D.B. (1984) Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases. J. Biol. Chem., 259, 15587-15596. Hartzell,H.C. and Titus,L. (1982) Effects of cholinergic and adrenergic agonists on phosphorylation of a 165 000-dalton myofibrillar protein
in intact cardiac muscle. J. Biol. Chem., 257, 2111-2120. Hofmann,P.A., Hartzell,H.C. and Moss,R.L. (1991) Alterations in
1959
M.Gautel et al. Ca2+sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J. Gen. Physiol., 97, 1141-1163. Holden,H.M., Ito,M., Hartshorne,I. and Rayment,I. (1992) X-ray structure determination of telokin, the C-terminal domain of myosin light chain kinase, at 2.8 A resolution. J. Mol. Biol., 227, 840-851. Jarcho,J.A., McKenna,W., Pare,J.A.P., Solomon,S.D., Holcombe,R.F., Dickie,S., Levi,T., Donis-Keller,H., Seidman,J.G. and Seidman,C.E. (1989) Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1. N. Engl. J. Med., 321, 1372-1378. Jeacocke,S. and England,P. (1980) Phosphorylation of a myofibrillar protein of Mr 150 000 in perfused rat heart, and the tentative identification of this as C-protein. FEBS Lett., 122, 129-132. Labeit,S., Gautel,M., Lakey,A. and Trinick,J. (1992) Towards a molecular understanding of titin. EMBO J., 11, 1711-1716. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. LeGrice,S.F.J. and Gruininger-Leitch,F. (1990) Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. Eur J. Biochem., 187, 307-314. Moos,C. and Feng,I.M. (1980) Effect of C-protein on actomyosin ATPase. Biochim. Biophys. Acta, 632, 14 1-149. Moos,C., Offer,G., Starr,R. and Bennett,P. (1975) Interaction of Cprotein with myosin, myosin rod and light meromyosin. J. Mol. Biol., 97, 1-9. Moos,C., Mason,C.M., Besterman,J.M., Feng,I.M. and Dubin,J.H. (1978) The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1. J. Mol. Biol., 124, 571-586. Musacchio,A., Saraste,M. and Wilmanns,M. (1994) High-resolution crystal structures of tyrosine kinase SH3 domains complexed with proline-rich peptides. Structural Biol., 1, 546-551. Nierendorf,R.C., Percy,C. and Young,R.A. (1987) Gene isolation by screening lgt 11 libraries with antibodies. Methods Enzvmol., 152, 458-469. Obinata,T., Reinach,F.C., Bader,D.M., Masaki,T., Kitani,S. and Fischman,D.A. (1984) Immunochemical analysis of C-protein isoform transitions during the development of chicken skeletal muscle. Devel. Biol., 101, 116-124. Offer,G., Moos,C. and Starr,R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. J. Mol. Biol., 74, 653-676. Okagaki,T., Weber,F.E., Fischman,D., Vaughan,K.T., Mikawa,T. and Reinach,F.C. (1993) The major myosin-binding domain of skeletal muscle MyBP-C (C-protein) resides in the COOH-terminal immunoglobulin C2 motif. J. Cell Biol., 123, 619-626. Pages,G., Brunet,A., L'Allemain,G. and Pouyssegur,J. (1994) Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK 1) EMBO J., 13, 3003-3010. Pearson,R.B. and Kemp,B.E. (1991) Protein phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol., 200, 62-81. Politou,A., Gautel,M., Pfuhl,M., Labeit,S. and Pastore,A. (1994a) Immunoglobulin-type domains of titin: same fold, different stability? Biochemistrv, 33, 4730-4737. Politou,A.S., Gautel,M., Joseph,C. and Pastore,A. (1994b) Immunoglobulin domains of titin are stabilized by N-terminal extension. FEBS Lett., 352, 27-31. Rasmussen,U.B., Basset,P. and Daniel,J.-Y. (1989) Direct amplification of cDNA inserts from I libraries using the cloning-adapter as primer for PCR. Nucleic Acids Res., 17, 3308. Rossi,E., Zarrilli,R. and Zuffardi,O. (1993) Regional assignment of the gene coding for a human Graves' disease autoantigen to 10q21.3q22. 1. Hum. Genet., 90, 653-654. Saiki,R.K., Scharf,S.J., Faloona,F., Mullis,G.T. and Erlich,H.A. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Scienice, 230, 1350-1354. Sanger,F., Nicklen,S. and Coulsen,A.R. (1977) DNA sequencing with chain inhibitors. Proc. Natl Acad. Sci. USA, 74, 5463-5467. Schiender, K. and Bean,L.J. (1991) Phosphorylation of chic cardiac Cprotein by calcium/calmodulin-dependent protein kinase 11. J. Biol.
Chem., 266, 2811-2817. Schultheiss,T., Lin,Z., Lu,M.-H., Murray,J., Fischman,D.A., Weber,K., Masaki, T., Imamura,M. and Holtzer,H. (1990) Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils. J. Cell Biol., 110, 1159-1172.
1960
Schweitzer,D. (1981) Counterstain-enhanced chromosome banding. Hum. Genet., 57, 1-14. Soteriou,A., Gamage,M. and Trinick,J. (1993) A survey of interactions made by the giant muscle protein titin. J. Cell Sci., 104, 119-123. Squire,J. (1981) The Structural Basis of Muscular Contraction. Plenum Press, New York and London, pp. 346-362. Starr,R. and Offer,G. (1978) The interaction of C-protein with heavy meromyosin and subfragment-2. Biochem. J., 171, 813-816. Starr,R. and Offer,G. (1982) Preparation of C-protein, H-protein, Xprotein and phosphofruktokinase. Methods Enzvmol., 85, 130-138. Studier,F.W., Rosenberg,A.H., Dunn,J.J. and Dubendorff,J.W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzvmol., 185, 60-89. ThierfelderL., Watkins,H., Macrae,C., Lamas,R., Mckenna,W., Vosberg,H.P., Seidman,J.G. and Seidman,C.E.(1994) a-Tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell, 77, 701-712. Thompson,J.D., Higgins,D.G. and Gibson,T.J. (1994) CLUSTAL-W: improving the sensitivity of progressive, multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res., 22, 4673-4680. Vaughan,K.T., Weber,F.E. and Fischman,D.A. (1992) cDNA cloning and sequence comparisons of human and chicken muscle C-protein and 86 kDa protein. Symp. Soc. Exp. Biol., 46, 167-177. Vaughan,K.T., Weber,F.E., Einheber,S. and Fischman,D.A. (1993) Molecular cloning of chicken myosin-binding protein H (MyBP) H (96 kDa protein) reveals extensive homology with the MyB-C (Cprotein) with conserved immunoglobulin C2 and fibronectin type III motifs. J. Biol. Chem., 268, 3670-3676. Vinkemeyer,U., Obermann,W. and Furst,D.O.(1993) The globular head domain of titin extends into the center of the sarcomeric M band. J. Cell Sci., 106, 319-330. Weber,F.E., Vaughan,K.T., Reinach,F.C. and Fischman,D.E. (1993) Complete sequence of human fast-type and slow-type muscle myosinbinding-protein C (MyBP-C). Eur J. Biochem., 216, 661-669. Williams,A.F. and Barclay,A.N. (1988) The immunoglobulin superfamily: domains for cell surface recognition. Annu. Rev. Immunol., 6, 381-405. Williams,A.F., Davis,S.J. and Barclay,A.N. (1989) Structural diversity in domains of the immunoglobulin superfamily. Cold Spring Harbor Symp. Quant. Biol., 54 , 637-647. Yamamoto,K. and Moos, C. (1983) The C-proteins of rabbit red, white, and cardiac muscles. J. Biol. Chem., 258, 8395-8401. Yanish-Perron,C., Vieira,J. and Messing,J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the MI 3mp 18 and pUC 19 vectors. Gene, 33, 103-119. Received on December 8, 1994; revised on Februarv 7, 1995
