Characterization of cDNA clones encoding a human fibroblast ...

Vol 266, No. 25, Issue of September 5, pp. 16917-16924,1391 Prrnted in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY Cc) 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization of cDNA Clones EncodingHuman a Fibroblast Caldesmon Isoform andAnalysis of Caldesmon Expressionin Normal and Transformed Cells* (Received for publication, January 9, 1991)

Robert E. Novy, Jenny Li-Chun Lin, and Jim Jung-Ching LinS From the Departmentof Biology, Uniuersity of Iowa, Iowa City, Iowa 52242

Overlapping cDNA clones encoding a low Mr human nonmuscle caldesmon isoform (HUM 1-CaD) span the entire coding region (538 amino acids) as well as 111 base pairs (bp) of 5’-noncoding and 1249 bp of 3’noncoding region. Northern blot probes derived from either the coding or 3“noncoding region hybridized to a 4.3-kilobase mRNA in nonmuscle cells and a 5.2kilobase mRNA in stomach tissue. Primer extension results indicated that the 5’-noncoding region of the HUM 1-CaD mRNA is approximately 700 bp in length and also suggested that 1-CaD mRNAs with common 5‘-noncoding regions are expressed in both liver and fibroblast cells. Comparisons of the human, rat, and chicken 1-CaD amino acids sequences demonstrated that although each isoform has unique characteristics, extensive regions of conservation exist. Amino acids 27-53 and 97-127 are 100% identical in these isoforms while amino acids 297-531 of HUM 1-CaD are 9 4 and 85% identical to the rat and chicken 1-CaDs, respectively. In addition, the levels of HUM 1-CaD mRNA and protein appeared to be decreased by 2-4 fold in the transformed derivatives of KD and W138 celllines as judged by Northern and Western blot analysis. The results suggest that the decrease of 1CaD protein in thesetransformed cells is a direct result of decreased 1-CaD mRNA synthesis and/or increased mRNA turnover.

fragments of caldesmon together with recentcDNA sequencing reports (5, 6, 19-21) has led to delineations of functional domains which are concentrated in the amino- and carboxylterminal regions of the protein. Binding domainsfor myosin (7-lo), calmodulin (16-18), actin (11-16), and tropomyosin (13, 19, 20) as well as potential phosphorylation sites (22-27) have been proposed. I n vitro, caldesmon exhibitsaninhibitory effect on the actomyosin ATPase by binding to F-actin(28). The presence of tropomyosin on F-actin isknown to potentiate thecaldesmon-mediated inhibition (1-3) while Ca*’/calmodulin attenuates the inhibition via a direct interaction with caldesmon (29-31). Phosphorylation of caldesmon has also been shown to release this ATPase inhibition (32) and in onecase correlates with reduced actin and calmodulin binding affinities (27). The inhibitory effect of caldesmon is believed to be mediated by displacing myosin fromactin (7),or by inhibiting the V,,, of ATP hydrolysis without weakening the myosinactin binding (33) or by a combination of these mechanisms whentropomyosinisboundtoF-actin (34).Acarboxylterminal fragmentof caldesmon has been characterized as the F-actin binding/myosin ATPase inhibitory domain (11-13, 17) while an amino-terminal fragment has beenshown to interact withmyosin (7-9). Since caldesmon’s myosin-binding domain is not requiredfor myosin ATPase inhibition, several investigatorshave proposed that it may serve to form an inactive ternary myosin-caldesmon-actin complex and thereby providea means of creatingthe“latch”state of smooth muscle contraction (7,35). Therefore,based on these Caldesmon (CaD)’ is a protein postulated to participate in analyses it is generally accepted that a thin filament-based the regulation of actin-myosin-basedcontractionjmotility regulation of actin-myosininteraction coexistsin smooth processes in smooth muscle and nonmuscle cells (reviewed in muscle and nonmuscle cells with the previouslyidentified Refs. 1-3). Early investigations revealed the existence of two thick filament-based regulatory system, i.e. phosphorylation My classes of caldesmon, high My (h-CaD) and low My (1of myosin light chain. CaD), which possessed common biochemical properties. The A variety of dataindicatesthat1-CaD playsa role in primary difference between these My classes is the absence of a highly repetitive central domain in thelow My class (4- nonmuscle motility processes and F-actin filament organiza6). Extensiveanalysis of chemical andproteolytic (7-18) tion. The following evidence was cited in a reveiw by Sobue et al. (1)in supportof this contention.1)Detailed examination *This workwas supported by grants HD18577, GM40580 and of 1-CaD colocalization with F-actin revealed a periodic disHL42266 from the National Institutes of Health. The costs of publi- tribution on stress fibers coincident with tropomyosin and cation of this article were defrayed in part by the payment of page complementary to a-actinin.2) I n vitro reconstitution expercharges. This article must therefore be hereby marked “aduertise- iments demonstrated that 1-CaD also inhibited the actinrnent” in accordance with 18 U.S.C. Section 1734 solely to indicate 3) Separate experiments suggested activated myosin ATPase. this fact. T h e nucleotide sequence($ reportedin this paperhas been submitted that 1-CaD may play a role in the motile processes of chromaffin granule secretion and concanavalin A receptor captotheGenBankTM/EMBLDataBankwith accessionnumber(s) M64110. ping. 4) 1-CaDwas found toregulate the interaction of the F$ T o whom allcorrespondence andreprint requestsshouldhe actin cross-linkingprotein filamin with actin i n vitro (see Ref. addressed. 1, and references therein). More recent i n vitro investigations ’ The abbreviations used are: CaD, caldesmon; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; nt, nucleotide(s); aa, have demonstrated that 1-CaD enhances the F-actin binding amino acid(s); TCP, total cell protein; TM, tropomyosin; TnT, tro- affinity of nonmuscle tropomyosins especially the low My ponin T. isoforms (36), 1-CaD potentiates tropomyosin inhibition of

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Human Fibroblast Caldesmon

theF-actin severing activity of gelsolin (37), and 1-CaD potentiates tropomyosins enhancement of F-actin annealing after F-actin is severed by gelsolin (38). Consistent with a motility-related function, we have found that microinjection of a monoclonal antibody (C21) recognizing a caldesmon epitope near the F-actin/calmodulin-binding domains results in severely decreased movement of intracellular granules (39). In addition, recent in uitro phosphorylation experiments have suggested that theobserved dissociation of RAT 1-CaD from microfilaments during mitosis (27) may be a consequence of mitotic-specific phosphorylation mediated by cdc2 kinase (21). Clearly, these results suggest that 1-CaD plays a significant role in the intricacies of nonmuscle microfilament dynamics. Therefore, investigation of 1-CaD structure andfunction should further elucidate the complex interactions of the nonmuscle cytoskeleton responsible for motile processes. In thispaper we report ahuman caldesmon cDNA sequence encoding a low My isoform (HUM 1-CaD) of 538 amino acids ( M r = 62,698). Northern blots with HUM1-CaD probes demonstrated the presence of distinct CaD mRNAs in human nonmuscle and smooth muscle cells. Comparisons of HUM 1CaD with the recently reported 1-CaDsequences from chicken brain (6), chicken oviducts (5), and rat liver (21) revealed highly conserved amino- and carboxyl-proximal regions. As was the case in the CHK 1-CaDs (5,6), the shorterlength of HUM 1-CaD is due to the absence of a central repetitive domain. In addition, HUM 1-CaD mRNA and protein levels in human cell lines were characterized by Northernand Western blot analysis, respectively. Direct comparisons of parental cell lines with their transformed derivatives demonstrated that decreased levels of HUM 1-CaD mRNA and protein were present in the latter. EXPERIMENTAL PROCEDURES

lip fibroblast cell line (American Type Culture Collection); HUT-11, a chemically transformed derivative of KD (gift from T. Kakunago, National Cancer Institute); CRL-1420, a human pancreatic carcinoma cell line; EJ, a human bladder carcinoma cell line; MCF-7, a human breast adenocarcinoma cell line (CRL-1420, EJ, and MCF-7 were all gifts from L.B. Ctan, Sidney Farber Cancer Institute, Harvard Medical School); WI-38, a human fetal lung diploid cell line (ATCC CCL75); and WI-38 VA13, a SV40 transformed derivative of WI-38 (ATCC CCL75.1). Allcells were grownin Dulbecco’s modified Eagle’s mediumcontaining 10% fetal calf serum and maintained in a 37 “C humidified incubator with 95% air and 5% CO,. Western Blot and SDS-Polyacrylamide Gel Electrophoresis-Cells grown on 100-mm dishes at 90% confluence were washed three times with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.5 mM KHzPO,, 8 mM Na2HP04, pH 7.3) and then solubilized in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer (2% SDS, 15%glycerol, 100 mM dithiothreitol, 80 mM Tris, pH 6.8,0.001% bromphenol blue) containing 1 mM phenylmethylsulfonyl chloride and 2 pg/ml leupeptin and immediately boiled for 3 min. Proteins in total homogenates were then resolved by 12.5% sodium dodecyl sulfate-polyacrylimide gel electrophoresis (40), and Western immunoblotting was performed as previously described (40). Northern Blot-Totalcell RNA was isolated by the guanidine isothiocyanate method (43). Northernblotting was performed as described (42). 8 pg of total cell RNA was loaded on a 1%formaldehyde/agarose gel. After electrophoresis the RNA was transferred to zetaprobe membranes (Bio-Rad) eletrophoretically using aTE52 transphor tank (Hoefer). DNA probes were labeled according to a radioactive PCR-labeling protocol (44). Sp6 and T, primers were used to generate the PCR-labeled probe. Hybridization and wash conditions suggested by the zetaprobe membrane manufacturer (Bio-Rad) were followed. Primer Extension-Primer extension was performed as described (42). 40pgof total RNA wasused as template. A 5’-noncoding, antisense oligonucleotide 30-mer corresponding to nt61-90 was used as primer. Sequence Alignments-Alignments of 3”noncoding regions and amino acid sequences were obtained by using the DNASTAR programs ALIGN and AALIGN, respectively.

Materials

RESULTS

Restriction enzymes and DNA modifying enzymes were purchased from Promega and New England Biolabs. Sequenase kits were purchased from United States Biochemical Corp. and Nick Translation and Erase A Base kits were purchased from Promega. PCR primers were purchased from the University of Iowa DNA Corefacility. Zeta probe membranes and nitrocellulose were purchased from Bio-Rad and Millipore, respectively. Hybond-N and Hybond-C membranes were purchased from Amersham Corp. or ICN. Common reagents were purchased from either Sigma or Fisher Scientific. Autoradiography was performed using X-OMAT AR film (Kodak).

cDNA Clones Encoding HUM 1 -CUD-Previous characterization of ouranti-chickenh-CaD monoclonal antibodies revealed that monoclonal C21 had broad-species caldesmon specificity including cross-reactivity with a putative human nonmuscle 1-CaD (40). Accordingly, C21 was used to screen a Xgtll expression library prepared from a human fetal lung fibroblast cell line, WI-38. To guard against mutations introduced during library construction/propagation and/or PCR amplification, the nucleotide sequence of at least three independent clones was determined for every coding region position (nt112-1726, Fig. 1).Although single base pair variability was noted at 10 positions, this did not appear to be the result

Methods

cDNAClone Isolation and Characterization-A Xgtll expression library prepared from WI-38 human fetal lung fibroblast poly(A)+ mRNA was purchased from Stratagene. Monoclonal antibody C21, previously characterized to recognize HUM 1-CaD (40), was used to screen the library following the procedures of Huynh et al. (41). After plaque purification, cDNA inserts of positive clones were PCR amplified by using primers which flanked the Xgtll EcoRI site. The cDNAs were subsequently cleaved with EcoRI and subcloned into the EcoRI site of pGEM3zf- (Promega). Plasmids were prepared for sequencing by the boiling method (42). Sequencing with Sequenase was performed using SP6 and T7 primers and a caldesmon-specific antisense primer corresponding to nt 1486-1513. Large clones were sequenced followingthe creation of a series of nested deletions (Erase A Base Kit, Promega). One clone, CaDl9, contained the majority of CAD39 the HUM 1-CaD-coding region (Fig. 1). A 5’-EcoRI fragment of CAD1 CaD19 (nt 516-840) was used as a DNA probe for a second round of FIG. 1. A composite restriction enzyme map of a HUM 1library screening resulting in theisolation of clone CaD35. A 5‘ XmnI fragment of CaD35 (nt 341-465) was in turn used in an additional CaDcDNA derived from independent overlapping cDNA screening which yielded clones (CaD 37, 38, and 40) extending into clones. A X g t l l cDNA expression library prepared from the human the 5’-noncoding region. The above probes were labeled by the nick fetal lung fibroblast cell line WI-38 was screened immunologically translation method (Nick Translation Kit, Promega) and hybridiza- with monoclonal antibody C21. Additional clones were obtained by using the 5”EcoRI fragment of CAD19 and the5’-XmnI fragment of tions were performed (42). Cell Culture-Cell lines used in this study included KD, a human CAD 35 as nick-translated probes.

Human Fibroblast Caldesmon of multiple 1-CaD genes based on the distribution of the mutations among the clones (data not shown). The concensus nucleotide sequence and the derived amino acid sequence of HUM 1-CaD is given in Fig. 2. A low M-y class of nonmuscle caldesmon538 amino acidslong is encoded by the concensus cDNA given that translation initiates at a Kozak (45) initiation site (ATG, nt112-114) and terminates at the first in-frame stop codon (nt 1,726-1,728). This protein has a calculated M-y of 62,698 and a PI = 6.2. In addition to ..nt

1

CAGATCATCAAATCAAATTCCACAGGGATTGGTGACCAACCAGAAGGCTCA

52 GACATCTGATTGCTGACCTGTCCAGACATCATCTGGTCTCCCTGAACCTGAAATCACACC aa

M D D F E R R R E L R R Q K R E E M R L 20 112 ATGGATGATTTTCAGCGTCGCAGAGAACTTAGAAGGCAAAAGAGGGAGGAGATGCGACTC E A E R I A Y Q R N D D D E E E A A R E OD 172 GAAGCAGAAAGAATCCCCTACCAGAGGAATGACGATGATGAAGAGGAGGCAGCCCGGGAA R R R R A R Q E R L R Q K Q E E E S L C 60 232 CGCCCCCGCCGAGCCCGACAGGAACGGCTGCGGCAGAAGCAGGAGGAAGAATCCTTGGGA Q V T D Q V E V N A Q N S V P D E E A K EO 292 CAGCTCACCGACCAGGTGGAGGTGAATGCCCAGAACAGTGTGCCTGACGAGGAGGCCAAG T T T T N T Q V E G D D E A A F L E R L l O O 352 ACAACCACCACAAACACTCAAGTGGAAGGGGATGATGAGGCCGCATTCCTGGAGCGCCTG A R R E E R R Q K R L Q E A L E R Q K E l 2 0 412 GCTCGGCGTGACGAAAGACGCCAAAAACGCCTTCAGGAGGCTCTGGAGCGGCAGAAGGAG F D P T I T D A S L S L P S R R H Q N D l 4 0 472 ~TCGACCCAACAATAACAGATGCAAGTCTGTCGCTCCCAAGCAGAAGAATGCAAAATGAC T A E N E T T E K E E K S E S R Q E R Y l 6 0 532 ACACCAGAAAATGAAACTACCGAGAAGGAAGAAAAAAGTGAAAGTCGCCAAGAAAGATAC E I E E T E T V T K S Y Q K N D W R D A l E O 592 GAGATAGAGGAAACAGAAACAGTCACCAAGTCCTACCAGAAGAATGATTGGAGGGATGCT E E N K K E D K E K E E E E E E K P K R 2 0 0 652 GAAGAAAACAAGAAAGAAGACAAGGAAAAGGAGGAGGAGGAAGAGGAGAAGCCAAAGCGA G S I G E N Q I K D E K I K K D K E P K 2 2 0 712 GGGAGCATTGGAGAAAATCAGATCAAAGATGAAAAGATTAAAAAGGACAWGAACCCAAA E E V K S F M D R K K G F T E V K S O N 2 4 0 772 GAAGAAGTTAAGAGCTTCATCCATCGAAAGAAGGCATTTACAGAAGTTAAGTCGCAGAAT G E F M T H K L K H T E N T F S R P G G 2 6 0 832 GGAGAATTCATGACCCACAAACTTAAACATACTGAGAATACTTTCAGCCGCCCTGGAGGG R A S V D T K E A E G A P Q H E A G K R 2 8 0 892 AGGGCCAGCGTGGACACCAAGGAGGCTGAGGGCGCCCCCCAGATGGAAGCCGGCAAAAGG L E E L R R R R G E T E S E E F E K L K 3 0 0 952 CTGGAGCAGCTTCGTCGTCGTCGCGGGGAGACCGAGAGCGAAGAGTTCGAGAAGCTCAW Q K Q Q E A A L E L E E L K K K R E E R 3 2 0 1012 CAGAACCAGCACGAGGCGGCTTTGGAGCTGGAGGAACTCAAG~GAGGGAGGAGAGA R K V L E E E E Q R R K Q E E A D R K L 3 4 0 1072 AGGAAGGTCCTGGAGGAGCAAGAGCAGAGGAGGAAGCAGGAGGAAGCCGATCGAAAACTC R E E E E K R R L K E E I E R R R A E A 3 6 0 1132 AGAGAGCAGCAACAGAAGAGGAGGCTAAAGGAAGAGATTGAAAGGCGAAGAGCAGAAGCT A E K R O K M P E D G L S D D K K P F K M O 1192 GCTGAGAAACGCCP;GAAGATGCCAGAAGATGGCTTGTCAGATGACAAGAAACCATTCAAG C F T P K G S S L K I E E R A E F L N K L O O 1252 TGTTTCACTCCTAAAGGTTCATCTCTCAAGATAGAAGAGCGAGCAGAATTTTTGAATAAG S V Q K S S G V K S T H Q A A I V S K I 4 2 0 1312 TCTGTGCAGAAAAGCAGTGGTGTCAAATCGACCCATCAAGCAGC~TAGTCTCCAAGATT D S R L E Q Y T S A I E G T K S A K P T 4 4 0 1372 GACAGCAGACTGGAGCAGTATACCAGTGCAATTGAGGGAACAAAAAGCGCAAAACCTACA K P A A S D L P V P A E G V R N I K S M 4 6 0 1432 AAGCCGGCAGCCTCGGATCTTCCTGTTCCTGCTGAAGGTGTACGCAACATCAAGAGTATG W E K G N V F S S P T A A C T P N K E T 4 8 0 1492 TGGGAGAAAGGGAATGTCTTTTCATCCCCCACTGCAGCAGGCACACCAAATAAGGAAACT A G L K V G V S S R I N E W L T K T P D 5 0 0 1552 GCTGGCTTGAAGCTAGGGCTTTCTAGCCGCATCAATGAATGGCTAACTAAAACCCCAGAT G N K S P A P K P S D L R P G D V S S K 5 2 0 1612 GGAAACAAGTCACCTGCTCCCAAACCTTCTGACTTGAGACCAGGAGACGTATCCAGCAAG R N L W E K Q S V D K V T S P T K V * 538 1672 CGGAACCTCTGGCAAAAGCAATCTGTGGATAAGGTCACTTCCCCCACTAAGGTTTGAGAC 1732 1792 1852 1912 1972 2032 2092 2152 2212 2272 2332 2392 2452 2512 2572 2632 2692 2752 2812 2872 2932

16919

the coding region 111bp of 5’- and 1249 bp of 3”noncoding were obtained. A potential polyadenylation signal AATAAA is present at nucleotide positions 2147-2152. However, this location is far removed from the 3’ terminus (nt 2, 975) of the cDNA characterized thus far. Presumably a second polyadenylation signal will be present near the3’-poly(A) tail. A previous report concerningchicken (CHK) CaD isoforms indicated thath and 1-CaDisoforms were encoded by mRNAs (4.8 and 4.1 kb, respectively) whichwere expressed in a tissuespecific manner (6). However, Bryan et al. (19) reported that CHK h-CaD was encoded by 4.1-kb (major transcript) and 3.5-kb (minor transcript) mRNAs. In order to determine the tissue specificity of expression and the RNA transcript size in humans, Northern blotsof total RNAisolated from human fibroblasts, liver, and stomach were probed with ”P-labeled CaD40 DNA (nt25-840). Fig. 3 clearly demonstrates that the nonmuscle cells/tissuewhich expressHUM1-CaD (fibroblasts and liver) transcribe a 4.3-kb CaD mRNA while the smooth muscle stomach tissue which expresses HUM h-CaD protein transcribes a 5.2-kb mRNA. When the 3’-noncoding region of CAD39 (nt 1,767-2,975) was usedas probeidentical results were observed (data not shown), thereby suggesting that HUMh and 1-CaDisoforms may be derived from a single gene via alternativesplicing. As noted above discrepancies exist between the reports of Hayashi et al. (6) and Bryanet al. (19) regarding CHK h-CaD mRNA size. Bryan et al. reported that their CHK h-CaD cDNA had a 3”noncoding region 1606 bp long. In contrast, Hayashi et al. reported a 3”noncoding region of only 55 bp for both CHK h-CaD (20) and CHK 1-CaD (6) and further suggested that the sequence ACTAA locatedimmediately upstream of 16 consecutive A nucleotides(the terminalnucleotides of their clone) served as thepolyadenylation signal. If true, thebulk of the mRNAs lengthwould, therefore, have to be present in the 5”noncoding regions. Thus, in order to define the natureof HUM 1-CaD mRNAs3‘- and 5’-noncoding regions, a combined analysis of primer extension and computer alignment were performed. The 3‘-noncoding regions of HUM 1-CaD and CHK h-CaD (19) were aligned using the DNASTAR Align program. As

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n

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AGTTCCACAAAGAACCCAAGCTCAAGACGCAGGACGAGCTCAGTTGTAGAGGGCTAATTC1791

GCTCTGTTTTGTATTTATGTTGATTTACTAAATTGGGTTCATTATCTTTTATTTTTCAAT1851 ATCCCAGTAAACCCATGTATATTATCACTATATTTAATAATCACAGTCTAGAGATGTTCA 1911 TGCTAAAAGTACTGCCTTTGCACAGGAGCCTGTTTCTAAAGAAACCCATGCTGTGAAATA 1971 GACACTTTTCTACTGATCATCATAACTCTCTATCTGAGCAGTGATACCAACCACATCTGA 2031 AGTCAACAGAAGATCCAAGTTTAAAATTGCCTGCGGAATGTCTCCAGTATCTAGAAAAAT 2091 GAACCGTAGTTTTTGTTTTTTTAAATACAGAAGTCATGTTGTTTCTGCACTTTATAATAA 2151 AGCATGGAAGAAATTATCTTAGTAGGCAATTGTAACACTTTTTGAAAGTAACCCATTTCA 2211 GATTTGAAATACTGCAATAATGGTTGTCTTT-GAAACGTACTGTTAA 2271 GGTATTACTTTTTTTCATGCTCATGATTCATATCTAAATTACATTATTATGTTAGCTGAC 2331 AGTGGTACTGATTTTTTACGTTGGTTGTTTTGTGGATTTCTTTAGTAGTGATAGTAGCCT 2391 GAACCACATTTTAGATAACTCAATTATGTATGTATGTGCATACACATATACAAACACACT 2451 AATGCTAGAATGCTTTTTTATGTGCTAGACTATTATATTTAGTAGTATGTCATTCTAACT 2511 AGCCAATATCACACCTTTTGAAAAATTAAAAAATCACACTATATTAATATTTCATATTTG 2571 CCAACAGAAACATGGCAGATAGGTATCAATATGTTTTCAATGCCTCATGACCTATAAGAA 2631 GAAAGTATTGAAAAGAAGACAGATTAGAACTGTTAGAAGGACTTGAAATTTTCTAAAAGA 2691 CATAGTATTTAGTTTATAATTAAATGCATTCTTGAAGTCCAGTGTCAATTTTATTAATGC 2751 TATCATCTCCACCAAGCTCAAAGCCTACTTATTAGAAACAATGAAGTTCACAATAGGTCA 2811 TAAGGTCTCTTCCTTTTCTAAAATTGAAAGACAAGAAATTTAGTGCCAATATTGTACAGA 2861 CAGAAATTCCATGTATGAGTCTCAACAAAGACTACCTTTGGCTAAATGTCTAGAAGCAGA 2931 GAAGTAAAGTGAGCAAAATCCAGTGTTGAGGAGTCATCCAATTC 2975

.D

5.2kbw 4.3kbw

rne2*’ 4

r

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-18s

*

I FIG.2. The concensus nucleotide sequence derived from overlapping cDNA clones as well as its deduced amino acid sequence of HUM 1-CaD cDNA. An open reading frame 538 amino FIG. 3. Northern blot analysis. 8 pg of total RNA isolated from acids long beginsa t a concensus Kozak (45) initiation sequence (Met, human kDcells, stomach, andliver were loaded in the lanesindicated. ?’P-Labeled CaD40 cDNA was used as probe. The probe hybridized nucleotides 112-114) and terminates a t a TGA stopcodon (nt 1,726liver and toa 5.2-kb mRNA band 1, 728). Encoded amino acids are indicated by the single letter code to a 4.3-kb mRNA band in KD and above their respective codons. The nucleotide and amino acids posi- in stomach. Identical results were obtained when the 3”noncoding tions areindicated by the columns on theleft and right, respectively. region of CaD39 cDNA (nt 1,767-2,975) was used as probe.

16920


can be seen in Fig. 4, extensive homologous regions exist between the two sequences. This result suggests that the extended 3' sequence reported by Bryan et al. (19) may represent the real 3"noncoding region in chicken. Thus, Hayashi et aL's (6, 20) truncated 3"noncoding regions may be the result of internal oligo(dT)primingduring cDNA svnthesis. Primer extension analysis was also performed to further define the HUM 1-CaD transcript. At present 111 bp of 5'noncoding and 2975 bp overall have been obtained from a transcript demonstrated to be approximately4.3 kb in length. In order to determine the extent of 5"noncoding region, an antisense oligonucleotide 30-mer corresponding to the 5'noncoding region nucleotides 61-90 was used to primer extend CaD mRNAs present in human liver and human fibroblasts (cell line WI-38) total RNA samples. The results shown in Fig. 5 reveal that thelongest major extension product terminates approximately 700 base 5' of the start codon in both WI-38 and liver RNA. Other major extension products were also observed terminating as doublets at nt 322-323 and nt 316-317 5' to the startcodon. These products may represent .reverse transcriptase pause sites or perhaps alternative transcription initiationsites. In anyevent, thesedata suggest that FIG.5. Primer extension study. A "P-end-labeled antisense HUM 1-CaD mRNAs with common 5"noncoding regions are 30-mer oligonucleotide corresponding to 5"noncoding region posipresent in boththe WI-38 and liver cells. It would also appear tions 61-90 was hybridized with 40 pg of total RNA from human liver that approximately 600 bp of 5'- and 600 bp of 3"noncoding or WI-38 fibroblasts. The primer was extended by reverse transcriptase and loaded on a 6%sequencing gel. Extension product sizes were region remains to be characterized. by comparison with sequencing ladders loaded in adjacent Amino Acid Sequence Comparison of HUM, RAT, and CHK determined lanes (not shown). I-CaDs-In addition to HUM fibroblast 1-CaD, several other 1-CaD cDNAs have recently been reported in rat liver (21), chicken brain (6), and chicken oviduct ( 5 ) . Amino acid se- CaD isoform was identical to theirpreviously described CHK quence alignment of these isoforms (Fig. 6) reveals that al- h-CaD except for an internal deletion spanning the central though there are extensive regions of conservation, each iso- repetitive region) (5). The NH2 terminus of CHK brain 1form possesses a unique combination of sequences in regards CaD is eight amino acids shorter than thatof CHK oviduct 1-CaD, and it hasa distinct NHz-terminal sequence (aal-16). to theamino acids which are inserted/deleted, present at the The NH2-terminal21 amino acids of HUM fibroblast 1-CaD NHz terminus and present at the COOH terminus. are identical to those reported for CHK oviduct 1-CaD while The greatest variation is observed in the amino-terminal the reported sequence of rat liver 1-CaD is 31 amino acids sequences. The CHK 1-CaD isoforms reported by Hayashi et longer (not shown) than eitherHUM 1-CaD or CHK oviduct al. (6) and Bryan et al. (5) differ only at theamino terminus 1-CaD. It is interesting to note, however, that aa43-60 of rat and one COOH-terminal position. (Note, the sequence data liver 1-CaD is similar to aal-16 of CHK brain 1-CaD. listed for CHK oviduct 1-CaD in Fig. 6 are based on a The sequence alignment also reveals substantial differences preliminary report in which they stated that their CHK 1- in the COOH-terminal coding region. Although RAT and HUM 1-CaDs are virtually identical inthis region, both v 2620v 2630v 2640v 2650v 2660v appear tobe truncated relative to theCHK 1-CaDs. The CHK CHK h-CaD TTTTGCATTATCCCAGTTAAAACCATGTATATTAGCACTATATTTAATAGT 1-CaDs are in turn identical with the exception of a single TTTT CA TATCCCAGT AAA CCATGTATATTA CACTATATTTAATA T HUM 1-CAD TTTTTCAATATCCCAGT-AAACCCATGTATATTATCACTATATTTAATAAT amino acid insertion in CHK brain 1-CaD (aa508). 1880^ 1890^ 1850* 1860^ 1870Other divergent and conserved internal coding regions are 2670v 2680v 2690v 2700v also notable. Both HUM and RAT 1-CaD have insertions of CHK h-CAD CAATCT----AGACATTCATCATAATAGTACTGCCTTTGCACAAGAACCT 7 (HUM 1-CaD aa81-87) and 26 (HUM 1-CaD aa271-296) CA T AGA TTCATTAA AGTACTGCCTTTGCACA GA CCT HUM 1-CAD CACAGTCTAGAGATGTTCATGGTAAAAGTACTGCCTTTGCACAGGAGCCT amino acids relative to the 1-CaDs in chicken. Overall, there 1900* 1910* 1920^ 19301940A appear to be three internal coding regions which are highly v 2720v 2730v 2740v 2750v conserved and separated by two regions which are more CHK h-CAD TTATTCCTAAAGAAACCCATGCTGTGAAATATACAGACTATCCTACTGAC variable. Two short regions of 100% identity corresponding T T CTAAAGAAACCCATGCTGTGAAATA AGACT T CTACTGA HUM 1-CAD GTTT--CTAAAGAAACCCATGCTGTGAAATAG--AGACTTTTCTACTGAT to HUM1-CaD positions aa27-53 and aa97-127 are separated 1950A 1960^ 1970^ 1980A by the more variable region aa54-96. The RAT 1-CaD isoform 2760v is 76% identical to HUM 1-CaD in this variable region while CHK h-CAD AGTCATAACT the CHK 1-CaDs are only 46% identical. Another variable TCATAACT HUM 1-CAD CATCATAACT region is found from HUM 1-CaD positions 128-296. Rat 11990^ CaD is 78% identical and the CHK 1-CaDs 44% identical, FIG.4. Comparison of the nucleotide sequences of the 3'- respectively, to HUM 1-CaD in this region. This is followed noncoding regions of HUM 1-CaD and CHK h-CaD. The 3'- by a third highly conserved region (aa297-531) in which RAT noncoding regions of HUM 1-CaD (nt 1, 726-2, 975) and CHK h- 1-CaD and the CHK 1-CaDs are 94 and 85% identical, reCaD (19) ( nt2, 503-4, 108) were aligned using the DNASTAR spectively, to HUM 1-CaD. A final notable difference in program ALIGN. An example of a highly homologous(79% identical) regard to future protein characterizations is in the number 156-bp region observed in the alignment is given here. The middle row designates identities between the two sequences. Dashes in the and distribution of Cys residues. All 1-CaDs have a cysteine residue corresponding to position aa381 in HUM 1-CaD. Only top and bottom rows indicate gaps.

Human Fibroblast Catdesmon HUMCAD RATLVCAD CHKOVCAD CHKBRCAD

HUMCAD RATLVCAD CHKOVCAD CHKBRCAD



350v 340v 330v 320v 310v HUMCAD EAALELEELKKKREERRKVLEEEEQRRKQEEADRKLREEEEKRRLKEEIER

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position aa145 in the CHK brain sequence. Expression of HUM I-CaD in Normal and Transformed Cells-Owada et al. (46) previously demonstrated that RAT 1-CaD protein levels were decreased approximately %fold in transformed cells relative to their normal parentalcell lines. Lin et al. (40) also noteda similar decrease in the amount of 1-CaD present in the immunoprecipitated microfilament fraction of a rat-transformed cell line. In this study,we have also found that HUM 1-CaD protein levels are decreased in transformed human cell lines. Fig. 7 demonstratestheresults obtained by Western blot analysis with the anti-CaDmonoclonal antibody C21. Total cell protein homogenates (TCP) were loaded so that at least twice as much transformed cell line T C P was applied relative to normal cell line T C P based on histone protein band intensities (Fig. 7 A ) . Fig. 7B demonstrates that the transformedderivatives of WI-38 and KD cell lines (WI-38VA13 and HUT-11, respectively) had a t least 2-fold lower levels of 1-CaD protein as judged by scanning densitometry of the autoradiograph. 1-CaD protein inMCF7 was detectable only upon prolonged exposures. A notable difference in 1-CaD protein levels between the normal cell lines KD and WI-38was also apparent. Morphologically, KD exhibits a flatter, more thoroughly spread appearance than the WI-38 cell line. The relevance of morphology to 1-CaD expression levels is, however, unknown at this time. In any case, direct comparisions of normal parental cells with their transformedderivatives revealed decreased 1-CaDprotein levels in the latter. In order to determine if this decrease inprotein levels corresponded to a decrease in 1-CaD mRNA levels, Northern blotanalysis wasperformed ontotal RNAisolatedfrom several normal and transformed cell lines including those examined in the Western blot analysis. Total RNA concentrations were determined usinga spectrophotometer and similar amountsof total RNA were loaded in each laneas can be seen from the ethidiumbromide staining (Fig. 8 A ) . Note that based on 28 S and 18 S rRNA band intensities lanes 4 (HUT11) and 6 ( WI-38 VA13) are loaded with greater or equal B A 1 2 3 4 5

1 2 3 4 5

kd 200116.5-

FIG. 6. Comparisons of the amino acid sequences of HUM I-CaD and other known 1-CaDs. The amino acid sequences of RAT liver (RATLV) 1-CaD (aa37-573) (21), CHK brain ( C H K B R ) 1-CaD (aal-517) (6), and CHK oviduct ( C H K O V ) 1-CaD (aal-524) (5) were aligned against HUM 1-CaD (aal-538). Dashes indicate identities, asterisks indicate gaps, and nonidentities are indicated by the single letter amino acid code relative to HUM 1-CaD atthat position. The numbers on the toprow correspond to HUM 1-CaD and the numbers on the bottom row correspond to CHKbrain 1-CaD. Note that the first 36 amino acids of RAT liver I-CaD are not included in this comparison.

FIG. 7. Western immunoblot analysis. Monoclonal antibody C21 was used in a Western blot analysis of total cell protein (TCP) to detect HUM 1-CaD protein levels. Lanes 1-7 wereloaded as follows: 1 , WI-38; 2, WI-38 VA13; 3, KD; 4, HUT-11; and 5, MCF-7. Approximately equal amounts of normal cell line TCP (WI-38 and KD) were loaded in lunes I and 3. At least twice as much transformed cell line TCP (WI-38VA13, HUT-11, and MCF-7) was loaded in lunes HUM 1-CaD lacks a n additional Cys. A second Cys residue 2 , 4 , and 5 ( A , sodium dodecyl sulfate-polyacrylamide gel electrophoin RATliver and CHK brain1-CaD is located in their distinctresis) based on histone protein staining intensity. Western blot analyamino-terminal regions. A third Cys in CHK brain 1-CaD sis ( B ) revealed that HUM 1-CaD protein levels were decreased in and a secondCys inCHK oviduct 1-CaD are located a t WI-38 VA13 and HUT-11 relative to WI-38 and KD, respectively.

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amounts of RNA as compared to lanes 5 (KD)and 7 ( WI38),respectively. The cDNA insert from CAD40 (Fig. 1) was used as probe, and the results are presented in Fig. 8B. The probe hybridized to a single 4.3-kb mRNA band in lanes 2-7. Identical results were obtained using the 3"noncoding region of CAD39 (nt 1,767-2,975) as probe (data notshown). Again, direct comparison of the normal fibroblast cell lines, KD and WI-38 (Fig. 8B, lanes 5 and 7) with their transformed derivatives, HUT-11 and WI-38 VA13 (lanes 4 and 6 ) revealed a t least 2-fold decreases in the latter's 1-CaD mRNA levels. A 4.3-kb mRNA band was detected in the MCF-7 sample only upon prolonged exposures (data not shown). Note that the relative levels of 1-CaD mRNA detected in Fig. 8 correlate well with protein levels detected inFig. 7. This result suggests that 1-CaD mRNA levels may be the primary determinant of 1-CaD protein expression. In order to demonstrate that general mRNA degradation was not a reasonable explanation for the decreased 1-CaD mRNA levels observed in transformed cells, a control Northern blot hybridization was performed. A labeled cDNA fragment of human y-nonmuscle actin cDNA (nt 191-704) (47) was used to probe a duplicate blot of that shown in Fig. 8B. The results shown in Fig. 8C argue against general mRNA degradation. The lanes loaded withtransformed cell total RNA (lanes2 , 3 , 4 , and 6 ) exhibit similar or greater amounts of y-actin mRNA as compared to thelanes loaded with normal cell total RNA (lanes5 and 7).Thus, when available for direct comparison 1-CaD mRNA levels in normal cells were shown to be substantially higher than in their transformed derivatives.

The amino terminus of RAT liver 1-CaD is very different from that of HUM fibroblast and CHK oviduct 1-CaDs. In humans, however, Northern blot and primer extension analyses indicate that both fibroblasts and liver express a 4.3-kb CaD mRNA with a common 5"noncoding region. This does not, however, preclude the possibility that the RNA samples contained multiple 1-CaD mRNAs nor does it preclude the possible existence of CaD mRNAs that have common 5'noncoding region exons connected to alternatively spliced NH2-terminalcoding region exons. Likewise, it remains to be determined if there are HUM 1-CaD isoforms which have elongated CHK 1-CaD-like COOH termini. Future RNA protection experiments using HUM 1-CaD sequences as probe will be required to clarify these possibilities. Comparisons of the HUM1-CaD amino acid sequence with those of CHK andRAT revealed extensive regions that were highly conserved and others thatwere widely divergent. Obviously, these similarities and differences may reflect evolutionary constraints imposed by the interaction of caldesmon with isoforms of myosin, actin, tropomyosin, Ca2+/calmodulin, and protein kinases. Numerous studies using either chemical/proteolytic fragments of purified native caldesmon (718) or using caldesmon deletion mutants expressed in Escherichia coli (6, 48) have delineated caldesmon's functional domains. The myosin-binding domain, for example, has been generally localized to theamino-terminal region of the protein (7-10). Thus, it is tempting to speculate that the short regions of 100% identity noted above may interact with regions conserved in the numerous myosin isoforms while the more variable neighboring regions may be involved in determining the isoform specificity of these interactions. DISCUSSION Likewise, the tropomyosin (TM) binding site(s) on caldesThe fact that two distinct 1-CaD isoforms have been re- mon remain only generally defined. Two potential TM-bindported inchicken raises the possibility that multiple, presum- ing sites were proposed based on sequence similarities of CHK ably tissue-specific 1-CaDs are also expressed in other species. h-CaD with the tropomyosin-binding proteintroponin T (TnT). One site originally proposed by Bryan et al. (19) (CHK h-CaD aa509-565) was similar to a region of TnT, aa90-146, characterized as part of TnTs Ca2+-insensitive TM-binding domain (49, 50). This region corresponds to aa310-366 of HUM 1-CaD, which contains identical alternating charge boxes as described by Bryan et al. (19). A second site originally proposed by Hayashi et al. (CHK h-CaD 622-636) (51) was kb -9.5 similar to a region of TnT, aa244-257, characterized as part -7.5 of TnTs Ca2+-sensitive TM-binding domain (49, 50). This ,.4.4 -Call corresponds to aa409-423 of HUM 1-CaD, which has three fewer identities with T n T relative to theCHK caldesmons in -2.4 this region, but two of those changes are conservative in *1.4 nature (ThrAla) (Fig. 9A). Using a computer database search, we have detected an additional CaD-TnT similarity which is presentedin Fig.9B. The 26 amino acid insert sequence -0.24 present only in HUM and RAT 1-CaD (HUM 1-CaD aa271296) contains a region aa280-293 which shares nine identities with rat cardiac T n T aa148-161 (52) and six identities with 3 4 5 6 7 rabbitskeletal T n T aa121-134 (50) (notethatthethree W b W r n "Y-Act in additional nonidentities are all conservative changes). The relevance of these CaD-TnT similarities to actual CaD-TM interactions, however, remains unclear. Studies employing FIG.8. Northern blot analysis of the HUM 1-CaD expres- caldesmon deletion mutants (6,48)have indicated that CHK sion in normal and transformed human cell lines. HUM 1-CaD cDNA clone CaD40 was used to probe total RNA samples isolated h-CaD residues aa509-565 are insufficient for strong CaDTM interaction. Rather, these studies suggest a more extenfrom normal and transformed human cell lines. 8 pg of total RNA were loaded in each lane. Lanes 1-7 were loaded as follows: 1, MCF- sive area of CaD-TM contactand/or multiple, separated 7; 2, CRL-1420; 3, EJ; 4, HUT-11; 5, KD;, 6, WI-38 VA13; and 7, WI- regions of contact. 38. A shows the total RNAloading as visualized by ethidium bromide The major F-actin- and Ca*+/calmodulin-binding domains staining. B shows the Northern blot results when CaD40 cDNA was on the other hand are more rigorously defined and have been used as probe. C shows the Northern blot results when a control hybridization was performed using y-actin cDNA (nt 191-704) (47) localized to a relatively small 10 kDa COOH-terminal CHK h-CaD fragment aa659-756 (corresponding to HUM 1-CaD as probe.

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Human Fibroblast Caldesmon A

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formed phenotype. The expression level of high My TM isoforms which have high F-actin affinity commonly decreases KXAGTTAK-GKVGGR while the level oflow My TM isoforms with weak F-actin RABBIT SKEL. TnT 244 affinity increases intransformed cells (55-59). Gelsolin IS’ XSTHQAAIVSKIDSR H U M 1-CaD expression levels decrease in transformed cells (60) and cor623 409 respondingly increases in transformation revertants (62). The expression of a-actin also decreases in transformed cells (63). B HUM I-CaD KRLEELRRRRGETES The level of Ca2+and calmodulin (64) and the level of a 5521p n3 kDa protein which inhibits TM binding to F-actin in uitro RAT CARDIAC NRLAEERARREEEES TnT 147 161 (65), all increase upon transformation. Owada et al. (46) and RABBIT this reportdemonstrated that the level of 1-CaD is also SKEL. TnT NRLAEEKARREEEDA decreased intransformed cells. 1-CaD has been shown to 120 1% FIG. 9. Amino acid sequence similarity between HUM 1- enhance microfilament stability in uitro by 1) enhancing the CaD and troponin T.A, HUM 1-CaD is included in a comparison F-actin affinity of nonmuscle tropomyosins (36) and 2) by originally presented by Hayashi et al. (51). A region of the Caz+- potentiating the TM-mediatedprotection of F-actin from sensitive tropomyosin-binding domainof rabbit skeletal T n T is com- gelsolins severing and capping activities (37, 38). Thus, depared with regions of HUM 1-CaD and CHK h-CaD having sequence creases in l-CaD levels combined with the perturbations similarities. B, sequences found within the HUM 1-CaD insert region noted above could result indecreased microfilament stability/ aa271-296 have sequence similarity with sequences found withinthe Ca2+-insensitive tropomyosin-binding domainof rat cardiac and rab- protection and altered microfilament composition. The severing and/or organizing activities of other F-actin-binding bit skeletal T n T (50, 52). proteins might then be able to mediate the drastic reorganiaa461-538) (16). This conclusion is also supported by results zation of microfilaments observed in transformed cells. obtained during our immunological screening of the cDNA REFERENCES library. The monoclonal antibody C21 has been shown to 1. Sobue, K., Kanda, K., Tonaka, K., and Uebi, N. (1988) J. Cell. inhibit CHK h-CaD binding to both F-actin and Ca2+/calBiochem. 37,317-325 modulin (53). Accordingly, C21 has also been shown to bind 2. Chalovich, J. M. (1988) Cell Biophysics 12, 73-85 the COOH-terminal 10-kDa fragment noted above (53). The 3. Bretscher, A. (1986) Nature 321, 726-727 4. Riseman, V. M., Lynch, W. P., Nefsky, B., and Bretscher, A. epitope of C21 was further refined in this study when it (1989) J. Biol. Chem. 264,2869-2875 recognized the P-ga1:HUM 1-CaD fusion protein produced by 5. Bryan, J., Saavedra-Alanis, V., Wang, C.-L. A., Wang, L.-W., and the Xgtll clone CaDl (data notshown). CaDl (Fig. 1)encodes Lu, R. C. (1990) J. MUSC.Res. Cell Motil. 11,434a aa487-538 of HUM 1-CaD which corresponds to aa685-736 6. Hayashi, K., Fujio, Y., Kato, I., and Sobue, K. (1991) J . Biol. in CHK h-CaD (19). Thus, a variety of complementary data Chem. 266,355-361 has shown that the major F-actin-and Ca2+/calmodulin- 7. Velaz, L., Ingraham, R. H., and Chalovich, J. M. (1990) J . Biol. binding sitesare located in a highly conserved region near the Chem. 265,2929-2934 8. Ikebe, M., and Reardon, S. (1988) J . Biol. Chem. 263,3055-3058 COOH terminus. In addition to possessing the F-actin- and Ca2+/calmodulin- 9. Sutherland, C., and Walsh, M. P. (1989) J. Biol. Chem. 2 6 4 , 578-583 binding sites, the highly conserved COOH-terminal 1/3 of 10. Hemric, M. E., and Chalovich, J. M. (1988) J . Biol. Chem. 2 6 3 , caldesmon is also the location of multiple phosphorylation 1878-1885 sites. In uitro analysis has demonstrated that Ca2+/calmodu- 11. Szpacenko, A., and Dabrowska, R. (1986) FEBS Lett. 202, 182lin-dependent protein kinase I1 (22, 25, 26), protein kinase C 186 (22-25), and cdc2 kinase (27) all are able to phosphorylate 12. Yazawa, M., Yagi, K., and Sobue, K. (1987) J. Biochem. (Tokyo) 102, 1065-1073 sites in this region. Allof the 1-CaDs in Fig. 6 have five 13. Fujii, T., Ozawa, J., Ogoma, Y., and Kondo, Y. (1988) J. Biochem. conserved putative cdc2 kinase phosphorylation sites (Ser/ (Tokyo) 1 0 4 , 734-737 Thr Pro) corresponding to HUM 1-CaD positions Thr-383, 14. Mornet, D., Audemard, E., and Deroncourt, J. (1988) Biochm. Ser-469, Thr-475, Thr-498, and Ser-504. Additional putative Biophys. Res. Commun. 154,564-571 cdc phosphorylation sites that are unique to RAT, conserved 15. Hurricane, M.-C., Cavadore, C., Audemard, E., and Mornet, D. (1990) FEBS Lett. 269, 185-188 in HUM and RAT, and conserved in CHK1-CaDs are present at HUM 1-CaD posiitons 255-256, Ser-534 and CHK 1-CaD 16. Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238 position Thr-231, respectively. Although, the in vivo signifi17. Takagi, T.,Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) cance of phosphorylation at any of these sites has not been J. Biochem. (Tokyo) 106, 778-783 definitively determined, a strong correlation has been dem- 18. Wang, C.-L. A., Wang, L.-W. C., and Lu, R. C. (1989) Biochem. onstrated between the properties of RAT 1-CaD phosphorylBiophys. Res. Commun. 1 6 2 , 746-752 ated in uitro by cdc2 kinase (21) and the properties of RAT 19. Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.G. (1989) J. Biol. Chem. 2 6 4 , 13873-13879 1-CaD phosphorylated in uiuo by a mitotic-specific kinase (9). Upon transformation, the stressfibers (microfilament bun- 20. Hayashi, K., Kanda, K., Kimizuka, F., Kato, I., and Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511 dles) indicative of well spread, anchorage-dependent normal 21. Yamashiro, S., Yamakita, Y., Hosoya, H., andMatsumura F. cells are dissembled and replaced with a diffuse meshwork of (1991) Nature 349, 169-172 microfilaments. Numerous authors (55-60) have suggested 22. Adam, L. P., Haeberle, J. R., and Hathaway, D. R. (1989) J . Biol. that this cytoskeletal rearrangement may play a role in the Chem. 264,7698-7703 progression towards and maintenance of the transformed 23. Litchfield, D.W., and Ball, E. H. (1987) J. Biol. Chem. 262, 8056-8060 phenotype. Felice et al. (61) have concluded that the loss of microfilament bundles which occurs during transformation 24. Tanaka, T., Ohta, H., Kanda, K., Tanaka, T., Hidaka, H., and Sobue, K. (1990) Eur. J . Biochem. 1 8 8 , 495-500 by pp60 u-src is due to a reorganization of polymeric actin 25. Hathaway, D. R., and Adam, L. P. (1990) J . Musc. Res. Cell Motil. rather than due to a net depolymerization. Interestingly, the 11,435a expression of a number of microfilament-associated proteins 26. Ikebe, M., and Reardon, S. (1990) J . Biol. Chem. 2 6 5 , 17607is perturbed concomitant withthe establishment of the trans17612 CHK h-CaD

KPAHTTAWSKIDSR

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I I I

I I I I I I I II

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