Identification in turkey gizzard of an acidic protein related to the C

Communication Vol. 264, No. 24, Issue of August 25, pp. 13971-13974.1989 THE JOURNAL OF BIOLOGICAL CHEMISTRY

0 1989 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Identification in Turkey Gizzard of an Acidic Protein Related to the C-terminal Portion of Smooth Muscle Myosin Light Chain Kinase*

(Received for publication, April 19, 1989)

Masaaki ItoS, Renata Dabrowskagll, Vince Guerriero, Jr.& and David J. HartshorneS From the $Department of Animal Sciences, University of Arizona, Tucson, Arizona 85721 and the §Department of Muscle Biochemistry, Nencki Znstitute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland

The isolation of an acidic protein, PI 4.5, that is abundant in turkey gizzard is described. Its apparent molecular weight measured by electrophoretic procedures is 24,000. This protein is phosphorylated by the catalytic subunit of the CAMP-dependent protein kinase and one phosphorylation site is indicated. From sequence determinations of tryptic peptides it is con- cluded that this protein is closely related to the C- terminal part of smooth muscle myosin light chain kinase. The initiation site for the protein is to the C- terminal side of the calmodulin-binding site. From the sequence data an estimated molecular weight is 18,000. This protein is expressed independently, as indicated by a blocked N terminus, and is probably the translation product of the 2.7-kilobase RNA detected previously in chicken gizzard (Guerriero, V., Jr., RUSSO, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381). Because of its putative origin as the C-terminal end of smooth muscle myosin light chain kinase, it is termed “telokin” (from a combination of kinase and the Greek telos, “end”).

Contractile activity in smooth muscle is controlled by the phosphorylation and dephosphorylation of myosin and the regulatory enzymes myosin light chain kinase (MLCK)’ and phosphatase (1). Dabrowska et al. (2) were the first to isolate MLCK from smooth muscle (chicken gizzard), and they showed that two components were essential for activity. The higher molecular weight component was the apoenzyme of MLCK and the smaller component was calmodulin (3). As a byproduct of the calmodulin purification procedure a second acidic protein was isolated that was not effective in activating MLCK activity (2). In this study this protein will be referred to as telokin.

* This work was supported in part by National Institutes of Health Grants HL 23615 and HL 20984. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll Supported in part by the Polish Academy of Sciences within the project CPBP 04.01.

The abbreviations used are: MLCK, myosin light chain kinase; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid SDS, so- dium dodecyl sulfate; kb, kilobase(s); HPLC, high performance liquid chromatography; DTT, dithiothreitol.

Among the many studies on MLCK a key contribution was the isolation of a cDNA encoding about 60% of the native gizzard enzyme (4). This facilitated the assignment of func- tional domains including the ATP-binding site (4), the calmodulin-binding site (4-6), and the pseudosubstrate sequence (5, 7, 8). A distinction between MLCK from skeletal and smooth muscles is that the calmodulin-binding site in the former ends essentially at the C terminus of the molecule (9, IO), whereas in MLCK from smooth muscle there are approximately 150 residues extending from the calmodulin-binding site to the C terminus. The function of this part of the molecule is not known. In addition it is established that the apoenzyme of MLCK can be phosphorylated by the CAMP- dependent protein kinase (11) a t serine 511 or 512 (4, 6) and serine 525 (12). (The assignment of amino acids follows that used by Guerriero et al. (4).) From studies on the proteolysis of MLCK (7, 11, 13, 14) it is apparent that the C-terminal portion of the molecule (i.e. that extension not present in the skeletal muscle molecule) can be removed by cleavage at a protease-sensitive site close to the C-terminal side of the calmodulin-binding site. Using a-chymotrypsin, such limited proteolysis cleaves between the two phosphorylation sites

A puzzling feature in the study of Guerriero et al. (4) was that the 2.1-kb DNA (to MLCK) hybridized to two sizes of RNA: a 5.5-kb RNA, considered to be the mRNA for MLCK, and a smaller 2.7-kb RNA thought to be homologous to the C-terminal part of the molecule. The possibility therefore was raised that the 2.7-kb mRNA might encode for an unknown protein that shared a domain in common with MLCK (4, 15). The abundance of the 2.7-kb RNA suggested that the encoded protein would be a significant fraction of total protein, but by Western blots (using a polyclonal antibody to MLCK) the translated product of the 2.7-kb RNA was not detected in either gizzard (4) or oviduct (15).

It was realized subsequently that by changing the blotting conditions a second antigen could be detected. This paper reports the identification of the second protein in chicken gizzard that cross-reacts with polyclonal antibodies against myosin light chain kinase. Characterization of this protein has established it as a major component of smooth muscle and identical to the original protein isolated by Dabrowska et al. (21, i.e. telokin. Partial amino acid sequence has demon- strated its similarity to the C-terminal region of gizzard MLCK and therefore it is probably the translation product encoded by the 2.7-kb mRNA.

(14).

MATERIALS AND METHODS

MLCK was isolated from frozen turkey gizzard (7). Calmodulin was isolated from beef testes (16). The catalytic subunit of CAMP- dependent protein kinase was prepared from fresh bovine heart (17). Polyclonal antibodies to MLCK and telokin were prepared as described previously (18).

Peptides of telokin were isolated as follows: Telokin (total 3 mg) was phosphorylated in 6 mM MgC12,O.l mM (Y-~’P]ATP, 30 mM Tris- HC1 (pH 7.5), 1 mg/ml telokin, 5 pg/ml catalytic subunit of CAMP- dependent protein kinase at 25 “C for 1 h. The extent of incorporation (16) was about 1 mol of P/mol telokin. The reaction was stopped by immersion in boiling H20 for 2 min, followed by centrifugation at 20,000 X g for 15 min. The supernatant was dialyzed against 50 mM NH4HC03 (pH 8.0). Aliquots (0.9 mg of telokin) were digested with trypsin (Sigma, type XIII) at a 1:lOO w/w ratio of trypsin:telokin for

13971

13972 Protein Related to the C-terminal Part of Myosin Light Chain Kinase 19 h at 25 "C. The reaction was stopped by addition of diisopropyl fluorophosphate to 1 mM. Subsequent isolation of peptides by HPLC was as described previously (19) using a C-18 reverse phase column (Brownlee Labs, Spheri-5 RP-18, 220 X 4.6 mm). Three fractions were collected AP1 eluted at 35% CH3CN, 0.1% trifluoroacetic acid (this peak contained 32P); AP2 at 40% CH3CN, 0.1% trifluoroacetic acid; AP3 at 30% CH3CN, 0.1% trifluoroacetic acid. These were freeze-dried and sequenced using a Beckman 890 M sequencer in the presence of 2 mg of polybrene. Fractions from the sequencer also were monitored for 3zP by liquid scintillation counting. Amino acid composition, performic acid oxidation, and sequence analyses were carried out by Dr. A. Smith (University of California, Davis).

Electrophoresis was carried out on 7.5-20% polyacrylamide gradient slab gels (20). Telokin stained characteristically of acidic proteins (blue) with Stains-all (Eastman Organic Chemicals), and this procedure (21) was useful in its identification. The procedure for Western blots was as described by Tsang et al. (22) with the important modification that a potassium phosphate blotting buffer was used (23). Cross-reactivity with the antibodies was detected as described by Ito et al. (24). Protein concentrations were measured by the bicinchoninic acid protein assay reagent (Pierce Chemical Co.).

The initial part of the procedure for the isolation of telokin is a modification of the earlier method (2) and is presented in this section. The chromatographic procedures are given under "Results."

Frozen turkey gizzard (300 g) was minced and homogenized (War- ing blender) with 4 volumes of 30 mM MgC12, 0.2 M KC1, 1 mM EGTA, 0.5 mM DTT, 30 mM Tris-HC1 (pH 7.5), 1 mM benzamidine, 0.1 mM diisopropyl fluorophosphate (buffer A), filtered through cheese cloth, centrifuged at 12,000 X g for 15 min, and the supernatant collected. The pellet was subject to 2 cycles of homogenization, with 3 and 2 volumes of buffer A, and centrifugation. To the combined supernatants solid ammonium sulfate was added to 50% saturation, the mixture was centrifuged at 12,000 X g for 15 min and the supernatant adjusted to 70% ammonium sulfate saturation. Following centrifugation at 12,000 X g for 15 min, the pellet was dissolved in 150 ml of 30 mM Tris-HC1 (pH 7.5), 0.2 mM DTT and dialyzed against this buffer. After centrifugation at 15,000 X g for 15 min, the supernatant was adjusted to pH 4.9 (on ice) with HCl and after 30 min was centrifuged at 12,000 X g for 15 min. The supernatant was neutralized with Tris base.

RESULTS AND DISCUSSION

Chromatography of Telokin-The isoelectric supernatant was applied to DEAE-FF (2.5 x 23 cm) equilibrated with 30 mM Tris-HC1 (pH 7.5), 0.2 mM DTT. After the non-retarded proteins were removed by washing with the equilibration buffer a linear gradient of 0-0.4 M NaCl (2 X 400 ml) was applied and fractions monitored for telokin using the criteria of electrophoretic mobility and staining blue with Stains-all (most proteins are stained red). The apparent molecular weight is 24,000, as judged by SDS-gel electrophoresis. Frac- tions were collected as shown in Fig. l.4. The following additions were made: (NHJ2SO4 to 2 M; CaClz to 1 mM; leupeptin to 10 pg/ml and diisopropyl fluorophosphate to 0.1 mM, prior to application to a phenyl-Sepharose column (2 X 14 cm) equilibrated with 2 M (NH4&304, 1 mM CaClZ, 30 mM Tris-HzS04 (pH 7.5), 0.2 mM DTT. The column was washed with this buffer and a linear gradient (2 X 120 ml) from 2 to 0 M (NH4)zS04 applied at a flow rate of 30 ml/h. The elution profile is shown in Fig. 1B and the major peak contained telokin (>98% homogeneous). From the gel profiles shown in Fig. 1 it is evident that the major purification step occurred on chromatography with DEAE-FF. By this procedure the yield of telokin was approximately 80 mg from 300 g of gizzard.

Evaluation of the content of telokin in the initial extract (or in whole muscle) is hampered by proximity to an abundant protein of M, - 23,000 (see Fig. l ) , possibly that reported by Lees-Miller et al. (25). In addition, telokin is not stained by Coomassie Blue with the intensity found in more basic proteins. From the yield of telokin a cellular concentration of approximately 15 p~ is calculated (assuming the H20 content to be 75% of wet weight and a molecular weight of 24,000).

4.0 A 0.4

FRACTION NUMBER r I

Z.OX--X, I

k, X.

1.5 - I I g 1.0 -

0'

N U

0.5 -

1 2 3 4 5 6

205

974 I I6

66

-" 45 - - "29 4

0.0 0 ' 20 ' +LLqhLAo.o 40 60 80

FRACTION NUMBER

FIG. 1. Purification of telokin. A, DEAE-FF (Pharmacia LKB Biotechnology Inc.) chromatography. Fractions containing telokin (hatched urea) collected. B, phenyl-Sepharose chromatography. SDS- polyacrylamide gel, lune 1, original extract in buffer A; lune 2, 50- 70% saturation (NH4)*S04 fraction; lune 3, isoelectric supernatant; lune 4, DEAE-FF fraction; lune 5, phenyl-Sepharose fraction; lune 6, molecular weight markers. Arrour indicates position of telokin.

0 I cn a 0 a 0 0

0 IO 20 I I 30 40

1 50

TIME (min) FIG. 2. Phosphorylation of telokin by the catalytic subunit

of CAMP-dependent protein kinase. Conditions were: 10 mM MgCIZ, 0.1 mM [-y-32P]ATP, 30 mM Tris-HC1 (pH 7.5), 0.4 mg/ml telokin, 4 pg/ml catalytic subunit, 25 "c. 0, 0.1 mM CaC12; 0, 1 mM EGTA; 0 , O . l mM CaC12, 0.6 mg/ml calmodulin.

This is obviously an underestimate, but nevertheless is con- siderably higher than the concentration of MLCK in gizzard, calculated to be 4.6 p~ (26).

An additional property of telokin is its heat stability. Te-

Protein Related to the C-terminal Part of Myosin Light Chain Kinase 13973

lokin remains soluble in 0.2 M KCI, 30 mM Tris-HC1 (pH 7.5), 0.2 mM DTT after immersion in boiling H20 for 12 min.

Phosphorylation of Telokin-Shown in Fig. 2 are time courses of phosphorylation of telokin by the catalytic subunit of the CAMP-dependent protein kinase and these indicate that telokin has a single phosphorylation site. In different experiments between 0.7 and 1.2 mol of P/mol telokin (M, - 24,000) were incorporated. (Assuming an M, of 18,000 (see below), maximum incorporation was 0.9 mol of P/mol telokin). Phosphorylation kinetics were not influenced by the absence of Ca2+ or by the presence of Ca2+ plus calmodulin. Telokin was not phosphorylated by protein kinase C (under conditions optimal for light chain phosphorylation).

I 2 3 4 5 6 7 8 9 I C 1112

- 205 - w- r. - - - 116 rslir - 66

0 .L -97.4

m - 45

- 29

- 20 - I7

FIG. 3. Western blots using polyclonal antibodies to MLCK (lanes 2-4) and to telokin (lanes 5-8). Lanes 9-12 show control protein staining of nitrocellulose by naphthol blue black. Lanes I , 5, and 9, gizzard extract in buffer A, lanes 2, 6, and IO, purified telokin; lanes 3, 7, and 11, a-chymotryptic hydrolysate of MLCK (0.4 mg/ml MLCK in 30 mM Tris-HC1 (pH 7.5) digested at 25 "C for 15 min with 1:lOO w/w ratio of a-chymotrypsin:MLCK. Reaction stopped by addition of diisopropyl fluorophosphate to 0.2 mM); lanes 4, 8, and 12, purified MLCK.

Comparison of Telokin with MLCK-The relationship between telokin and MLCK was initially detected using Western blot and polyclonal antibodies to both MLCK and telokin. The blotting conditions are important, and telokin is retained by nitrocellulose only when the SDS concentration is reduced ( i e . conditions similar to those employed for Western blots of calmodulin (23)). In Fig. 3 it is shown that the antibody to MLCK recognized both the isolated telokin and a fragment of MLCK liberated by limited a-chymotryptic hydrolysis. I t is known that this fragment is the C-terminal part of MLCK (7, 11, 13-15). In addition it is shown in Fig. 3 that the antibody to telokin crossreacts with the following: the native MLCK and telokin in a crude extract of gizzard; isolated telokin; purified native MLCK and the C-terminal fragment released by proteolysis. However, the anti-telokin antibody did not crossreact with the larger a-chymotryptic fragment of MLCK. These results suggested a relationship between telokin and the C-terminal part of MLCK.

Other procedures were employed to investigate the similarity of the two proteins. By two-dimensional gel electrophoresis it was found that the mobility of telokin and the MLCK fragment were identical. The PI value was estimated to be 4.5. Proteolysis by Staphyloccus aureus protease gave identical patterns for the peptides generated from each protein. The amino acid composition of telokin was as predicted from the cDNA sequence of the C-terminal fragment assuming cleavage between residues 512 or 513 and 525. Cleavage in this region, i.e. to the C-terminal side of the calmodulin-binding site, is consistent with the finding that telokin did not bind to a calmodulin affinity column in the presence of Ca2+.

The most conclusive evidence indicating similarity of the two proteins is derived from sequence data. Direct sequence analysis of telokin was not possible because of a blocked N terminus. Tryptic peptides, therefore, were isolated and their sequence determined (see "Materials and Methods"). The results are shown in Table I in comparison with the sequence of MLCK deduced from cDNA. The phosphorylated peptide, AP1, had a sequence identical with residues 523-540 (the complete sequence was not determined and since amino acid analysis indicated the presence of tyrosine the peptide prob-

TABLE I Comparison of sequences of MLCK and peptides isolated from telokin

Sequences are from the cDNA of MLCK (4). The underlined sequences are those determined for the designated peptides. Potential initiation sites are indicated by ATG. In the sequence of the minor component of AP2, residues 613 and 614 were not identified, shown by dashed line.

513 520 530 ATG ATG A n ; M - A - M - I - S - G - M - S - G - R - K - A - S - G - S - S - P - T - S -

540 550 P - I - N - A - N - K - V - E - N - E - D - A - F - L - E - E - V - A - E -

560 E - K - P - H - V - K - P - Y - F - T - K - T - I - L - D - M - E - V - V -

AP2 570 580 E - G - S - A - A - R - F - D - C - K - I - E - G - Y - P - D - P - E - V -

AP3 590 600

M - W - Y - K - D - D - Q - P - V - K - E - S - R - H - P - Q - I - D - Y - AP2

610 620 D - E - E - G - N - - C - - - S - - L - T - I - S - E - V - C - G - D - D - D - A -

630 640 K - Y - T - C - K - A - V - N - S - L - G - E - A - T - C - T - A - E - L -

650 660 L - V - E - T - M - G - K - E - G - E - G - E - G - E - G - E - E - D - E - E - E - E - E - E

13974 Protein Related to the C-terminal Part of Myosin Light Chain Kinase

ably terminated at lysine 561). The phosphorylation site corresponds to serine 525 in agreement with earlier results (12). Evaluation of the cDNA sequence shows that there is only one likely consensus phosphorylation site for the CAMP- dependent protein kinase. The peptide AP2 was not homogeneous and its major and minor components gave sequences corresponding to residues 562-574 and 602-617, respectively. The third fraction, AP3, yielded the sequence 580-591. These data provide strong evidence that telokin and the C-terminal region of MLCK are closely related and provide a rationale for the term “telokin” (telos = end; plus kinase).

The initiation site of telokin is not known but could be at methionines 513, 515, or 519 (see Table I). The methionine content was determined as 4 mol/mol, by performic acid oxidation, and this suggests that the N-terminal amino acid is isoleucine 516 (assuming that after initiation methionine 515 is not incorporated into the protein structure). Telokin would therefore overlap with the C-terminal region of MLCK and be composed of 154 amino acids (see Table I). Its molecular weight would be about 18,000. The M , determined by SDS-gel electrophoresis, both for isolated telokin and for the MLCK fragment, clearly is anomalously high.

The sequence of telokin has no obvious homology to any other protein, except MLCK, as determined by a computer search of the National Biomedical Research Foundation Pro- tein Identification Resource databank using the FASTA pro- gram (27). Recently in a preliminary communication Shattuck et al. (28) have identified a protein in chicken gizzard presum- ably identical to telokin, which they suggest to be a calcium- binding protein, based on the 45Ca overlay technique. Previ- ously an acidic protein of M , 24,000 was identified in homog- enates of chicken gizzard using the same technique (29). The acidic PI and staining with Stains-all of telokin are character- istic of calcium-binding proteins but this must be validated by determination of binding parameters. Since telokin does not possess an EF hand structure (sequence screened using the oligonucleotide consensus sequence (30) and by comparison of the amino acid sequences of “classical” EF hands with telokin) high affinity binding may not be expected. Although there is no sequence homology between telokin and the mem- bers of the calpactin family (31) we have not eliminated the possibility that Ca2+-binding parameters may be modified by interaction with other molecules, or by phosphorylation.

The finding that telokin is closely related to the C terminus of myosin light chain kinase leads to speculation on the genomic organization of these two proteins. The most con- servative situation would be a single gene under the control of two promoters, rather than two independent genes. Regu- lation of each message by different promoters is supported by hormonal regulation of the message for telokin but not MLCK in oviduct (15) and the higher message levels for telokin in comparison to MLCK in gizzard (4). To our knowledge, this is the first example of a portion of a protein that can be independently expressed. I t will be interesting to compare the tissue distribution of telokin, since in skeletal muscle it is known that the C terminus of MLCK ends at the calmodulin-

binding site and the molecule does not contain the extension equivalent to telokin. Whether telokin is found only in those cells regulated by myosin phosphorylation is an intriguing question and is currently being investigated.

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Identification in turkey gizzard of an acidic protein related to the C