THE JOURNAL OF BIOLOGICAL CHEM~STRY

Printed in U S A. Vol. 258, No. 7, Issue of April 10, pp. 4539-4547, 1983

Analytical Subcellular Distribution of Calmodulin and Calmodulin- binding Proteins in Normal and Virus-transformed Fibroblasts*

(Received for publication, July 2, 1982)

Linda J. Van Eldik and Wilson H. Burgess From the Laboratory of Cellular and Molecular Physiology, Howard Hughes Medical Institute and Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232

We report a quantitative subcellular localization study of calmodulin and the subcellular distribution of calmodulin-binding proteins in normal and virus-trans- formed chicken embryo fibroblasts. We developed ho- mogenization conditions for fibroblasts which give maximal cell disruption with minimal organelle de- struction, prepared and characterized subcellular frac- tions, and measured the levels and distribution of cal- modulin in the fractions. We found that the majority of calmodulin is present in the soluble fraction, but that a small, reproducible amount of calmodulin is present in each of the particulate fractions. The amount of cal- modulin in all subcellular fractions of transformed fi- broblasts is greater than or equal to the amount of calmodulin in the corresponding fractions of normal fibroblasts. In contrast to the mostly soluble distribu- tion of calmodulin, many of the calmodulin-binding proteins in fibroblasts are associated with the particu- late fractions. Although there are no apparent quali- tative differences between normal and transformed fi- broblasts with respect to the number, distribution, or apparent molecular weight of the calmodulin-binding proteins, there may be quantitative changes in the levels of some calmodulin-binding proteins after trans- formation. These data suggest that many of the cal- modulin-binding proteins may be particulate proteins rather than soluble proteins, and that several calmod- ulin-binding proteins in fibroblasts may bind calmod- ulin in a calcium-independent manner.

Calcium is involved in the regulation of a variety of cellular processes. These processes appear to be mediated by fluxes in calcium concentration within the cell. The control by calcium of certain cellular events appears to be altered during tumor- igenesis or upon transformation of cells in uitro (1). The molecular mechanisms by which calcium fluxes bring about cellular responses and how the transformed cell escapes the normal regulatory controls are not understood.

The ability of chicken embryo fibroblasts to be transformed by avian RNA tumor viruses provides a well characterized cell system where transformation is directly related to tumor- igenesis (for review, see Ref. 2). In addition, cells are cultured only a few generations in vitro and exhibit more “normal” characteristics than do many permanent cell lines. Therefore, this biological system is one in which comparison of the relationships among calcium, calcium-binding proteins, and

* These studies were supported in part by funds from National Institutes of Health Grant GM30861. 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.

calcium-dependent cellular responses in normal and trans- formed cells can be made.

Calmodulin is an intracellular calcium-binding protein that is found in all eukaryotic cells examined, has a highly con- served amino acid sequence, and mediates multiple calcium- dependent activities in vitro (3-5). The physiological signifi- cance of most of these activities has not been demonstrated. Nevertheless, these multiple activities and ubiquitous distri- bution suggest that calmodulin plays an important role in calcium-mediated cellular responses. In addition, some of the cellular properties that are altered upon transformation may be regulated by calmodulin or calmodulin-binding proteins.

It has been shown previously (6, 7) that the freely soluble levels of calmodulin are increased in transformed CEF.’ The increased soluble levels of calmodulin are not the result of virus infection alone since cells that are treated with a repli- cation-competent, but transformation-defective mutant of Rous sarcoma virus do not show a comparable increase in calmodulin levels (6). The calmodulin molecule itself does not appear to be altered upon transformation of CEF since cai- modulin isolated from transformed CEF is indistinguishable from normal cell calmodulin by a number of physical and functional criteria (8).

In order to determine the subcellular distribution of cal- modulin and calmodulin-binding proteins and to gain insight into the mechanisms by which calmodulin homeostasis is altered in transformed cells, we used quantitative biochemical cytology techniques to measure the levels and distribution of calmodulin and calmodulin-binding proteins in normal and transformed CEF. These studies demonstrate that the total levels of calmodulin are increased in transformed CEF com- pared to normal CEF, suggest that the small amounts of calmodulin associated with particulate fractions may be func- tionally significant, and suggest that the levels of some cal- modulin-binding proteins may be altered after transformation.

EXPERIMENTAL PROCEDURES’

RESULTS

Preparation and Characterization of Subcellular Frat- tions-A necessary first step in these studies was definition of

’ The abbreviations used are: CEF, chicken embryo fibroblasts; EGTA, ethylene glycol bis(P-amino ethyl ether)-N,N,N’,N’-tetraa- cetic acid; SDS, sodium dodecyl sulfate; TEMED, N,N,N’,N’-tetra- rnethylethylenediamine.

Portions of this paper (including “Experimental Procedures” and Figs. 1-3 and 6 ) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 RockviUe Pike, Bethesda, MD 20814. Request Doc- ument No. 82M-1779, cite authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

4539

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4540 Calmodulin and Calrnodulin- binding Proteins in Fibroblasts

quantitative fractionation procedures for normal and trans- formed CEF. We used 0.25 M sucrose as an homogenization medium. We found that addition of calcium to the Sucrose resulted in aggregation of the subcellular particles during centrifugation, precluding quantitative analysis. This aggre- gation did not occur using 0.25 M sucrose alone or 0.25 M sucrose containing 1 mM EGTA. We found no differences in the subcellular distribution of calmodulin or calmodulin-bind- ing proteins when homogenizations were done in the presence or absence of 1 mM EGTA. Microscopic examination of CEF homogenized in 0.25 M sucrose using 8 strokes of the tight pestle of a Dounce homogenizer indicated that greater than 90% of the cells were disrupted. Assay of N-acetyl-P-glucosa- minidase activity showed lysosomal latencies of 70% or more for the homogenates. Transformed CEF were more easily disrupted than normal CEF, but retained latencies of 70% or more after 8 strokes. Therefore, these conditions were taken as optimal for maximal cell disruption with minimal organelle disruption. In all experiments, both normal and transformed CEF were homogenized under the same conditions.

Romogenates were fractionated by differential centrifuga- tion as described under “Experimental Procedures.” These conditions result in three particulate fractions (N, ML, and P) and a soluble fraction (S). The definition of subcellular frac- tions of CEF as nuclear (N), mitochondrial/lysosomal (ML), microsomal (P), and soluble (S) is an operational one based on precedents in other cell systems (12,26-28) and on results presented here. Subcellular organelles are differentially en- riched in the various fractions, and in this analytical study the different components of the fractions were not purified fur- ther.

Identical conditions were used for both normal and trans- formed CEF homogenates and the resulting subcellular frac- tions were characterized and compared. Electron microscopy of the particulate fractions from normal and transformed CEF showed that subcellular organelles are enriched in the appro- priate fraction and that the general distribution of organelles in each fraction is similar in both normal and transformed CEF.3 Subcellular fractions were also characterized by assay of various “marker” enzymes that have been reported to be predominantly associated with specific organelles. Some of the enzymes have previously been used in subcellular frac- tionation experiments (12, 26, 28) to yield characteristic dis- tribution patterns. Fig. 1 is a compilation of the data obtained for several fractionations and shows the distribution of these enzymes in the subcellular fractions of CEF. The results are expressed according to the representation initially proposed by deDuve et al. (26) as the relative specific activity (the percentage of the enzyme activity divided by the percentage of the protein) of the fractions plotted against their relative protein content. The distribution patterns of the marker en- zymes in CEF are similar to those obtained in other fibroblast systems (12, 28). In addition, the general distribution of all the enzymes is similar between normal and transformed CEF, although a larger percentage of the total protein is associated with the S fraction in transformed CEF.

Calmodulin Levels in Subcellular Fractions-We meas- ured the levels of calmodulin in each fraction from normal and transformed CEF using subcellular fractions prepared under the homogenization and fractionation conditions de- scribed above. Fig. 2 is a compilation of the data obtained from five separate subcellular fractionation experiments using phosphodiesterase activator activity as a measure of calmod- ulin. The majority (77-93%) of the total calmodulin is found

Electron micrographs of the particulate subcellular fractions are shown in Fig. 6 in the Miniprint.

in the s fraction in both normal and transformed CEF. The relative specific activity of calmodulin is at least four times greater in the S fractions compared to any of the particulate fractions (Fig. 2a). In addition, calmodulin levels are increased 2- to 3-fold in the S fraction of transformed CEF compared to the levels in the S fraction of normal CEF (Fig. 2b). Fig. 2b also shows that the calmodulin levels in all the particulate fractions of transformed CEF are equal to or greater than those in the particulate fractions of normal CEF. These data indicate that there is not a redistribution of calmodulin from particulate to soluble fractions in transformed CEF that could account for the increased soluble calmodulin levels in trans- formed CEF. These data also suggest that the total levels of calmodulin are increased in transformed CEF compared to normal.

It is possible that there may be additional calmodulin present that is not being detected by the phosphodiesterase activator assay (4,29,30). To directly address this possibility, subcellular fractions were boiled as described under “Experimental Procedures” and the boiled supernatants as- sayed for the presence of calmodulin. Fig. 3 shows the distri- bution and levels of calmodulin in the boiled fractions, as assayed by phosphodiesterase activator activity. Calmodulin is still found primarily in the S fraction and its concentration is approximately 3-fold higher in the S fraction from trans- formed CEF than in the S fraction from normal CEF. All the boiled subcellular fractions from transformed CEF showed calmodulin levels equal to or greater than the levels in the fractions from normal CEF. These data are further evidence that the total levels of calmodulin are increased in transformed CEF compared to normal CEF. The amount of calmodulin/ pg of protein is slightly higher in the boiled N and S fractions compared to the unboiled fractions but the increase is small and is not statistically significant.

Calmodulin levels in subcellular fractions were also deter- mined by radioimmunoassay using well characterized calmod- ulin-specific antisera (20) that detect calmodulin when it is free in solution and when it is part of a supramolecular complex (4). By radioimmunoassay, the distribution of cal- modulin in subcellular fractions of CEF was similar to that obtained using phosphodiesterase activator activity. The ma- jority (80-90%) of the calmodulin was found in the soluble fractions, and all the subcellular fractions from transformed CEF showed calmodulin levels equal to or greater than those in normal CEF. Table I gives a summary of the soluble calmodulin levels determined by both procedures. The specific activity of calmodulin determined by radioimmunoassay was less than that determined by phosphodiesterase activator activity, but by either procedure the increased calmoddin levels in transformed CEF versus normal CEF were signifi- cant. The amount of calmodulin detected in the boiled sub- cellular fractions was slightly greater than that in unboiled fractions whether radioimmunoassay or phosphodiesterase activator assay was used, but the difference between boiled and unboiled samples was not significant.

We have also examined the calmodulin levels as a function of cell number. When the calmodulin levels are expressed on a per cell basis, the differences between normal and trans- formed CEF are even greater than when the calmodulin levels are expressed/mg of protein.

Calmodulin-binding Proteins in Subcellular Fractions- Calmodulin is predominantly a soluble protein in CEF under the homogenization and fractionation conditions used. How- ever, there is a small, reproducible percentage of the total calmodulin found associated with the particulate fractions. It is not possible to determine the significance of the particulate localization of calmodulin in CEF using our subcellular frac-

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Calmodulin and Calmodulin-binding Proteins in Fibroblasts 454 1

TABLE I Calmodulin levels in normal and transformed CEF

Data are the mean 2 standard deviation. Numbers in parentheses represent the number of S fractions analyzed for calmodulin levels.

Phosphodiesterase activator activity Radioimmunoaswy

Unhoiled S fraction Roiled S fraction llnhoiled S fraction Boiled S fraction

np calmodulin/W: protein np ralmodulin/lyl protein Normal CEF 1.02 f 0.26 (8)" 1.55 f 0.21 (5) 0.88 2 0.30 (7)" 1.05 2 0.20 (8) Transformed CEF 3.08 f 1.29 (6)" 4.60 f 1.15 (5)' 1.51 f 0.23 (7)" 1.92 f 0.51 (8Ih

" Significantly different from each other ( p < 0.01). 'Significantly different from each other ( p < 0.001).

a

116 94 67

4 3

b

" "

A B C D E F G H I

C

c

" " A B C D E F G H I

d 94 67 43 3 0 -*

'%e

A B C D E F G H I

" .

2 0 -- 14 c

0.

A B C D E F G H I FIG. 4. The binding of '2SI-labeled calmodulin in the presence of calcium to proteins from the

subcellular fractions of normal and transformed CEF. In each panel, lanes A , B, C, and D contain the N, ML, P, and S fractions, respectively, from normal CEF. Lane E contains molecular weight standards (M, = 200,000, 116,000, 94,000, 67,000, 43,000, 30,000, 20,000, and 14,000). Lanes F, G, H, and I contain the N, ML, P, and S fractions, respectively, from transformed CEF. a and c show the Coomassie blue-stained 7.5% and 12.5% acrylamide- SDS gels, respectively. b and d show the corresponding autoradiographs depicting the binding of 't511-labeled calmodulin to discrete proteins.

tionation data alone because the percentage of the total cal- modulin associated with the particulate fraction is less than or equal to the percentage of the soluble marker enzyme activity (phosphoglucomutase) associated with these frac- tions. In order to determine whether the calmodulin associ- ated with the particulate fractions may represent a specific localization, we assayed the subcellular fractions for the pres- ence of calmodulin-binding proteins using a gel-binding pro-

cedure. The procedure takes advantage of the ability of known calmodulin-binding proteins to renature sufficiently in a gel matrix after electrophoresis in the presence of SDS to regain calmodulin-binding activity.

We have used the gel-binding procedure to estimate the number of calmodulin-binding polypeptides in the particulate and the soluble fractions of normal and transformed CEF. The results of one such experiment are shown in Fig. 4. a and

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4542 Calmodulin and Calmodulin- binding Proteins in Fibroblasts

c show Coomassie blue-stained 7.5% and 12.5% (w/v) acryl- amide-SDS gels to which equal amounts of protein from each subcellular fraction were applied. The amount of protein loaded onto the gels accounts for approximately 0.150%, 0.012%, 0.030%, and 0.014% of the total protein in the N, ML, P, and S fractions, respectively. b and d show the correspond- ing autoradiograms depicting the binding of "'I-labeled cal- modulin in the presence of calcium to discrete protein bands. Calmodulin-binding proteins can be detected in all of the subcellular fractions from both normal and transformed CEF. There are no major qualitative differences between normal and transformed CEF with respect to the number, distribu- tion, or apparent molecular weights of these proteins. How- ever, as judged by the intensities of the bands in the autora- diograms in Fig. 4, there may be quantitative differences in the levels of some calmodulin-binding proteins after transfor- mation.

Fig. 5 shows the results of "'I-labeled calmodulin-binding experiments done in the presence of excess unlabeled calmod- ulin (500-fold molar excess over labeled calmodulin) or in the presence of 5.0 mM EDTA in place of 1.0 mM CaCI.). a and c show the Coomassie blue-stained 12.5% (w/v) acrylamide- SDS gels which were incubated with "'I-labeled calmodulin in the presence of excess unlabeled calmodulin or EDTA, respectively. b and d show the corresponding autoradiograms depicting the binding of "'I-labeled calmodulin to the proteins in subcellular fractions from both normal and transformed CEF. As can be seen, an excess of unlabeled calmodulin competes with the '"I-labeled calmodulin for binding to the proteins on the gel. In addition, "'I-labeled calmodulin bind- ing to many but not all of the proteins is blocked by the addition of chelator. The majority of the proteins which bind "'I-labeled calmodulin in the presence of EDTA are found in the particulate fractions.

a P4"U

A B C D E

C

b

t 43

3 0

20

14

F G H I A B C D E F G H I

d 94 6 7 43

m -

A B C D E F G H I

30

20

14

A B C D E F G H I FIG. 5. The binding of '*%labeled calmodulin in the presence of unlabeled calmodulin or EDTA to

proteins from the subcellular fractions of normal and transformed CEF. In each panel, the lanes contain the same fractions as those shown in Fig. 4. The Coomassie blue-stained 12.5% acrylamide-SDS gels shown in a and c were incubated with ""I-labeled calmodulin under the same conditions as those shown in Fig. 4 with the exception that unlabeled calmodulin was added (a) , or EDTA was substituted for CaClr in the incubation buffer (c). b and d show the corresponding autoradiographs depicting the binding of "'I-labeled calmodulin to discrete proteins.

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Calmodulin and Calmodulin- binding Proteins in Fibroblasts 4543

DISCUSSION In this study we have I) defined conditions for reproducible

analytical subcellular fractionation of normal and virus-trans- formed CEF and have done a quantitative subcellular locdi- zation study of calmodulin in these cells; 2 ) demonstrated that the total levels of calmodulin are increased in transformed CEF compared to normal CEF, and that 77-93% of the cal- moddin in CEF is found in the soluble fraction; 3) detected the presence of calmodulin-binding proteins in both soluble and particulate subcellular fractions of CEF, and have found no apparent qualitative differences between normal and trans- formed CEF with respect to the number or apparent molecular weights of these proteins; and 4) demonstrated that there is no major redistribution of calmodulin, calmodulin-binding proteins, or marker enzymes upon transformation of CEF.

The data reported here confirm previous reports (6,7) that the freely soluble levels of calmodulin are 2- to 4-fold higher in transformed CEF compared to normal CEF, and demon- strate the validity of using soluble calmodulin as an estimate of total calmodulin activity in CEF. In this study, the in- creased calmodulin levels were demonstrated by both phos- phodiesterase activator assay and radioimmunoassay. It was important to measure calmodulin levels by more than one criterion. Measurements of biological activity may yield mis- leading results if other activators (31-34) or inhibitors (35,36) are present. Measurements of immunoreactivity also have limitations for determination of calmodulin levels. Immuno- reactivity of a protein does not necessarily correlate with biological activity of the protein (37), the immunoreactive sites of the protein may not be accessible to antibody ( i e . masking), or other proteins may cross-react with the antisera (38, 39). The antiserum used in these studies reacts with a unique heptapeptide in the COOH-terminal domain of verte- brate calmodulin and appears to bind calmodulin even when it is part of supramolecular complexes (4). Therefore, the potential problem of immunogenic masking or cross-reactivity is minimized. Even though the absolute values for the amount of calmodulin vary with the method of measurement, com- parison of the calmodulin levels in normal and transformed CEF by three independent procedures demonstrates a 2- to 4- fold increase in calmodulin levels in transformed CEF.

It has been reported (6, 40, 41) that calmodulin levels may vary depending on the density, proliferation state, or growth conditions of the cell. The culture conditions described under “Experimental Procedures” (harvesting of normal CEF 48-72 h after the last feeding) were chosen to render the normal CEF relatively quiescent and thus maximize the differences in calmodulin levels between normal and transformed CEF. However, we have also determined that there are increased levels of calmodulin in transformed CEF when compared to rapidly growing normal CEF. In addition, when normal and transformed CEF were maintained in culture and fed with media on the same time schedule, increased calmodulin levels in transformed CEF were still observed.

The increase in calmodulin levels does not simply reflect a general increase in all soluble proteins upon transformation. We have demonstrated (42) that the higher levels of calmod- ulin are mainly due to a selective increase in the rate of synthesis of calmodulin above that of other soluble and of total proteins. In addition, when the calmodulin levels are expressed on a per cell basis, the differences between normal and transformed CEF are even greater than when the cal- modulin levels are expressed per mg of protein. Thus, we have demonstrated, using various culture conditions and multiple assays for measurement, that the total calmodulin levels are higher in transformed CEF compared to normal CEF.

It has been reported (43-45) that in some cells and tissues,

the distribution of calmodulin between supernatant and pellet fractions varies with extraction conditions, e.g. the presence or absence of divalent cations. However, because marker enzymes, degree of organelle integrity, and extent of cell lysis were not measured, it is difficult to correlate supernatant and pellet fractions with soluble and particulate fractions as de- fined by biochemical cytology. The distribution of calmodulin and calmodulin-binding proteins between soluble and partic- ulate fractions of CEF was not changed significantly when homogenizations were done in the presence or absence of 1 m~ EGTA, Thus, in CEF, the majority of the calmodulin appears to be readily soluble under the various homogeniza- tion conditions used in this study. In addition, using biochem- ical cytology criteria, there does not appear to be any signifi- cant redistribution of calmodulin after transformation. This does not preclude the possibility that using other experimental approaches, such as qualitative immunocytochemistry, one could detect redistribution of a portion of the calmodulin molecules after transformation.

In contrast to the mostly soluble distribution of calmodulin in CEF, many of the calmodulin-binding proteins are associ- ated with the particulate fractions. The presence of these calmodulin-binding proteins in the particulate fractions is not a result of contamination by soluble proteins since many of the binding proteins show discrete localization to the partic- ulate or to the soluble fractions. In addition, efforts were made to minimize redistribution of proteins, such as during homog- enization or processing (46-48) by using carefully defined cell lysis and subcellular fractionation procedures. Finally, cal- modulin-binding proteins can be extracted from the ML and P fractions using urea or detergents and the extracted proteins do not remain soluble after removal of the chaotropic agents, suggesting they may be membrane-associated proteins.

It is also possible that the identification of so many calmod- ulin-binding proteins in the particulate fractions by our gel- binding assay reflects an increased stability of these proteins in the presence of SDS during electrophoresis. Biochemical information concerning the relative stability of calmodulin- binding proteins purified from membrane and soluble frac- tions is lacking so this question cannot be answered a t this time. The identification of calmodulin-binding proteins in particulate fractions is not due to interactions with membrane proteins in general because we see calmodulin binding to a relatively small population of all the proteins in the membrane fractions of CEF. Biochemical experiments of calmodulin in the calcium-saturated form uersus the calcium-depleted form are often interpreted to mean that hydrophobic domains on calmodulin are either exposed or buried upon addition of calcium. If this is true and the interaction of calmodulin with membrane proteins is simply a nonspecific, hydrophobic in- teraction, one would expect to see major differences in cal- modulin binding to membrane proteins in the presence of calcium or chelators. In contrast, our studies have shown that many of the calmodulin-binding proteins in the particulate fractions interact with calmodulin whether the incubation mixtures contain excess calcium or chelator. We conclude, therefore, that the interaction of calmodulin with the mem- brane proteins cannot be explained solely by nonspecific in- teractions with hydrophobic proteins. Our results suggest that the presence of calmodulin and calmodulin-binding proteins associated with particulate fractions may be physiologically relevant.

The results presented here are representative of 10 separate gel-binding assays on subcellular fractions. Although there do not appear to be any qualitative changes in the number or distribution of calmodulin-binding proteins upon transforma- tion, it is likely that there are quantitative changes in the

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4544 Calmodulin and Calmodulin- binding Proteins in Fibroblasts

levels of certain calmodulin-binding proteins. For example, we have demonstrated (42) that the activity of a calmodulin- regulated enzyme, myosin light chain kinase, is higher in extracts of transformed CEF compared to normal CEF. How- ever, information on the quantitative levels of calmodulin- binding proteins or estimates of their affinities for calmodulin cannot be obtained using gel-binding assays. It will be neces- sary to extend these initial observations to include purifica- tion, identification, characterization, and quantitation of these proteins.

The data presented here do raise the question of why the majority of calmodulin is found in the S fraction when such a large number of calmodulin-binding proteins are localized in particulate fractions. It should be noted that the free calmod- ulin levels are not known for any cell type. More importantly, it is not known for any cell type whether a significant amount of calmodulin ever exists in a “free” state in the cell. The concept of “soluble” levels is an operational one whose defi- nition depends on the experimental conditions being used. In properly executed biochemical cytology studies, the majority of the calmodulin is localized to the soluble or S fraction. This soluble fraction should not be equated with an intracellular, “free-floating” pool of calmodulin. Clearly, proteins localized to the soluble fraction in biochemical cytology studies could be integral components of supramolecular structures in the highly organized interior of a cell.

Calmodulin homeostasis has been reported to be altered in many transformed cells and tumor tissues (6, 7, 41, 42, 45, 49, 50). It is clear from the data summarized in this report that calmodulin regulation and its alteration in disease states is more complicated than initially suspected. Similarly, transfor- mation of cells is a complex process and is defined by multiple criteria. These criteria of transformation appear at different times after virus infection. Because of the complexities in the definition of when transformation occurs in cells and because of the inherent limitations of the assays it is not yet possible to correlate the changes in levels of calmodulin and calmod- ulin-binding proteins with molecular mechanisms of transfor- mation. Our direct demonstration (42) of a calmodulin-stim- ulatable myosin light chain kinase in CEF and an increase in myosin light chain kinase activity in transformed CEF that have increased calmodulin levels suggest that the cellular contractile apparatus may be one target of altered calmodulin regulation. Based solely on the known calmodulin-regulated enzymes in other biological systems, there are several other potential calmodulin-regulated activities, such as ion trans- port and metabolism, that might be altered in transformed CEF. It is clear that analysis of this pathophysiological system has generated the necessary tools and information for future studies on the identification and quantitation of calmodulin- binding proteins and for further investigation into mechanisms of calmodulin regulation in this and other biological systems.

Acknowledgments-We thank P. Lazarow and S. Fowler for help- ful advice on subcellular fractionation procedures. We are grateful to D. M. Watterson for his continued encouragement, stimulating dis- cussions, and critical reading of the manuscript.

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4546 Calmodulin and Calmodulin- binding Proteins in Fibroblasts

SUPPLEMENTARY MATERIAL TO

ANALYTICAL SUBCELLULAR O l S T R l B U T l O N OF CALMOOULIN AND CALMOOULIN BINDING PROTEINS

I N NORMAL AND VIRUS-TRANSFORMED FIBROBLASTS

L l n d a J . Van E l d l k a n d Wilson H. Burgess

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L J Van Eldik and W H Burgessin normal and virus-transformed fibroblasts.

Analytical subcellular distribution of calmodulin and calmodulin-binding proteins

1983, 258:4539-4547.J. Biol. Chem. 

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