©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Variation in the Expression and/or Phosphorylation of the Human Low Molecular Weight Stress Protein during in Vitro Cell Differentiation (*)

(Received for publication, November 8, 1994)

George Minowada (§) William Welch (¶)

From the Departments of Medicine and Physiology, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Members of the low molecular weight heat shock protein (hsp) family show regulated expression in both Drosophila and mice during development and differentiation. Here we have examined whether similar regulation of the single low molecular weight hsp (hsp 28) of humans exhibits differences in either its expression and/or phosphorylation during the course of in vitro differentiation of hematopoietic cells. In the promyelocytic leukemic cell line, HL-60, we show that early after commitment of the cells to a macrophage-like phenotype (via exposure to phorbol ester myristate, PMA) there occurs an accompanying increased phosphorylation of hsp 28. Over time and as the cells become terminally differentiated the levels of hsp 28 increase significantly. In contrast, cells stimulated to adopt a granulocyte-like phenotype (e.g. exposed to either dimethyl sulfoxide or retinoic acid) show no changes in either the phosphorylation or expression of hsp 28. Moreover, once differentiated the granulocyte-like cells no longer appear capable of phosphorylating hsp 28. Human K562 cells, in response to hemin, rapidly increase their expression and phosphorylation of hsp 28 during the course of their differentiation into erythroid-like cells. Addition of PMA to the K562 cells induces differentiation into a megakaryocyte-like phenotype but is not accompanied by changes in hsp 28 phosphorylation/expression. In the case of the HL-60 cells, differentiation toward the macrophage like lineage is accompanied by an increased adherence of the cells to their substratum and an apparent association of hsp 28 with the actin cytoskeleton.


INTRODUCTION

Most members of the heat shock or stress protein family are constitutively expressed proteins whose synthesis increases significantly in cells exposed to different environmental insults. Such an increase in the expression of the stress proteins appears to provide the traumatized cell with an added degree of protection, thereby contributing to the cell's overall ability to survive the particular environmental insult(1) . For at least some of the stress proteins, in particular the so-called hsp (^1)70 and hsp 60 families, recent evidence has implicated their role in facilitating the early events of protein maturation. Members of these two families, now also referred to more generally as molecular chaperones, interact with proteins which are in the course of synthesis, folding, and/or assembly into higher ordered structures. Via such an interaction the maturing polypeptide is temporarily provided a somewhat shielded environment thereby reducing the possibility of nonproductive intra- or intermolecular interactions which might occur otherwise during the course of its maturation (reviewed in Refs. 2, 3).

Relative to our new insights regarding the structure-function of those stress proteins which function as molecular chaperones, less is known regarding the function of the so-called low molecular weight heat shock proteins (low molecular weight hsps). Members of this family, with apparent molecular masses of 20-30 kDa, exist in vivo as higher ordered structures of 600-900 kDa(4) . At least four related members of this family have been described for insects like Drosophila(5, 6) . However. yeast, avian, and animal cells express only a single form of the protein, referred to by different investigators as hsp 25, hsp 27, or hsp 28 (the latter designation which will be used here)(7, 8, 9) . All of the members of this family exhibit sequence homology with the alpha-crystallin proteins, which like hsp 28, exist as higher ordered oligomeric structures in vivo(10) . While the exact function of both the low molecular weight hsps and alpha-crystallins is still unclear, both groups of proteins have been reported to function as molecular chaperones in vitro, facilitating both the refolding of denatured proteins and/or the stabilization of proteins in response to thermal treatments(11, 12, 13, 14) . In contrast to the hsp 70 and hsp 60 chaperones, however, such chaperoning activity provided by the low molecular weight hsps appeared independent of an added energy source such as ATP. Perhaps our best insight regarding potential function is a study reporting that the low molecular weight hsp purified from avian sources can both inhibit the polymerization of actin filaments as well as sever pre-existing actin filaments using in vitro assays(15) . Following up on this observation Landry and colleagues(16, 17) reported that animal cells overexpressing the low molecular weight hsp, via transfection of the corresponding cDNA, exhibited changes in their actin microfilament cytoskeleton and acquired a thermotolerant phenotype.

Expression of the low molecular weight hsps has been observed to vary as a function of development and/or differentiation in Drosophila and mice(18, 19, 20, 21) . In both yeast and mammalian tissue culture lines, expression of the low molecular weight hsp increases as the cell progress into their stationary growth phase, and in the case of yeast, during sporulation (22, 23, 24) . (^2)In addition to changes in its expression, changes in the phosphorylation of hsp 28 occur in response to a wide number of stimuli, some of which are associated with growth and/or differentiation (reviewed in (25) ). For example, hsp 28 is heavily phosphorylated within minutes after cells have been exposed to different mitogens, tumor promoters such as phorbol esters, calcium ionophores, or cytokines(26, 27, 28, 29) . In addition, the protein is heavily phosphorylated in cells subjected to heat shock and certain other metabolic stressors(26, 30) . Recent studies concluded that phosphorylation results in a reduction in the native size of the protein(31, 32) .

More than 10 years ago it was shown that an early event accompanying phorbol ester myristate (PMA) induced in vitro differentiation of promyelocytic HL-60 cells was a rapid increased phosphorylation of two proteins with apparent molecular masses of 17 and 27 kDa(33) . Subsequent work by the same investigators concluded that such phosphorylation of these two proteins was transient and likely dependent upon the calcium- and phospholipid-dependent protein kinase C(34) . Subsequently, it was reported that heat shock treatment also appeared to result in the initiation of HL-60 cell differentiation (35) . Because both heat shock and PMA result in a rapid increase in the phosphorylation of hsp 28, we undertook a careful study of the pattern of protein phosphorylation changes which accompany the differentiation of HL-60 cells in vitro. Here, we show that the 27-kDa protein that is rapidly phosphorylated during the early stages of PMA-induced differentiation of HL-60 cells into a macrophage-like phenotype indeed is hsp 28. Prompted by these observations we examined for possible changes in either the expression and/or phosphorylation of hsp 28 in other cell types capable of in vitro differentiation. We show that changes in hsp 28 expression/phosphorylation vary significantly as a function of both the cell type and the differentiation agent employed.


EXPERIMENTAL PROCEDURES

Cell Culture and Metabolic Labeling

HL-60 and K562 cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 200 units/ml of penicillin, and 200 µg/ml of streptomycin. HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, penicillin, and streptomycin. HL-60 cells were induced to differentiate into macrophage-like cells by the addition of phorbol myristate acetate to the media (PMA, 200 ng/ml; Sigma) for 4 h. The drug was then removed, and cells were incubated for an additional 68 h in fresh media without added PMA. To induce HL-60 cells to differentiate into a granulocyte-like cell, cells were grown in media supplemented with either dimethyl sulfoxide (Me(2)SO, 140 µM; Aldrich) or retinoic acid (RA, 1 µM; Sigma) for 72 h. K562 cells were induced to differentiate into megakaryocyte-like cells by growing in media supplemented with PMA (100 ng/ml) for 72 h. K562 cells were induced to differentiate into erythroid-like cells by growing in media supplemented with hemin (30 µM; Sigma) for 24 h.

For metabolic labeling, cells were washed in leucine-free RPMI and then incubated with [^3H]leucine (Amersham Corp.; specific activity 75 Ci/mM) in leucine-free RPMI supplemented with 2% fetal calf serum. Alternatively, cells were washed in phosphate-free RPMI or DMEM, incubated with [P] or [P]orthophosphate (Amersham; carrier-free, 10 mCi/ml) in phosphate-free RPMI or DMEM for 1 h, and then subjected to various treatments as described in the figure legends. After labeling, cells were washed in phosphate-buffered saline (PBS), solubilized in Laemmli sample buffer (LSB), heated to 100 °C for 5 min, and the labeled proteins analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis (PAGE). For two-dimensional SDS-PAGE, cell lysates were digested with DNase I and RNase A (each 10 mg/ml), quickly frozen, and then lyophilized. Samples were redissolved in isoelectric focusing buffer containing 9.9 M urea, 4% Nonidet P-40, 2% ampholytes, and 100 mM dithiothreitol.

Electrophoresis

One- and two-dimensional SDS-PAGE were done as described previously(36) . SDS-PAGE slab gels of 12.5% acrylamide concentration were used to resolve proteins. Isoelectric focusing gels employed a mixture of pH 5-7 (70%) and pH 3-10 (30%) Ampholines (Pharmacia). Resolved proteins were visualized by staining with Coomassie Blue or in the case of radiolabeled proteins, by autoradiography or fluorography.

Immunological Procedures

Immunoprecipitations of radiolabeled proteins from cell lysates were performed as described previously(36) . Immunoprecipitations were programmed with an equal amount of radioactive protein, as first determined by trichloroacetic acid precipitation and scintillation counting. The Peterson modified Lowry procedure was used to quantify total protein concentration in the cell lysates(41) . Western blotting of one-dimensional SDS-PAGE gels was performed as described by Towbin et al.(37) . Primary antibodies used included: (a) anti-hsp 28 rabbit polyclonal (38) ; (b) anti-hsp 28 mouse monoclonal, D5 (gift of R. J. B. King, 39); (c) anti-hsp 72/73 mouse monoclonal, N27(40) ; (d) anti-hsp 72 mouse monoclonal, C92(40) . Horseradish peroxidase-conjugated secondary antibodies were obtained from Cappel. Immunoreactive proteins were visualized using the enhanced chemilluminescence system (ECL; Amersham). Indirect immunofluorescence was performed as described previously(40) . Briefly, cells were grown on glass coverslips, fixed in 3.7% formalin (Fisher) diluted in PBS for 15 min, and permeablilized in PBS containing 0.1% Triton for 2 min. Monoclonal anti-hsp28 antibody was diluted 1:1000 in PBS supplemented with 1% bovine serum albumin (Sigma). Fluorescein-conjugated goat anti-mouse antibody (Cappel) was diluted 1:100 in PBS, 1% bovine serum albumin. Staining for actin was done with rhodamine-conjugated phalloidin. Coverslips were mounted using Gelvatol (Monsanto) and air-dried.


RESULTS

Changes in the phosphorylation of hsp 28 were examined in HL-60 cells during their early commitment to the macrophage-like phenotype following exposure to PMA. Cells were incubated with [P]orthophosphate and then exposed to 100 ng/ml of PMA for 30 min. As a control, the cells also were subjected to either a 44 °C heat shock treatment, or exposure to sodium arsenite, two potent inducers of the stress response and both known to stimulate the rapid increased phosphorylation of hsp 28 in other cell types(26, 30) . Following the different treatments, cell lysates were prepared and analyzed either by high resolution two-dimensional gel electrophoresis or immunoprecipitation using an antibody specific for hsp 28. The two-dimensional gel analysis revealed the previously reported(33, 34) increased phosphorylation of two proteins of 17 and 27 kDa. The 27-kDa protein phosphorylated after PMA treatment migrated in the two-dimensional gels in a position identical with that of hsp 28 (data not shown). That the 27-kDa protein which exhibited increased phosphorylation following PMA treatment was in fact hsp 28 was confirmed by immunoprecipitation from the cell lysates using an antibody specific for hsp 28. As is shown in Fig. 1, exposure to PMA, like heat shock or sodium arsenite treatment, resulted in a marked increase in the phosphorylation of hsp 28.


Figure 1: The 27-kDa protein rapidly phosphorylated in HL-60 cells following PMA addition is hsp 28. HL-60 cells growing at 37 °C were incubated with [32P]-H3PO4 for 1 h in DMEM lacking phosphate. The cells then were: maintained at 37 °C (control); subjected to a 44 °C heat shock; exposed to 100 ng/ml PMA; treated with 200 µM sodium arsenite; or 1 mM dibutyl cAMP. After 30 min the cells were harvested by the addition of Laemmli sample buffer and the lysates immediately heated at 95 °C for 5 min. Immunoprecipitations using a mouse monoclonal anti-hsp 28 antibody were performed and the resultant immunoprecipitates analyzed by SDS-PAGE. Shown is an autoradiograph of the gel. 37 °C control cells (lane 1); heat shock-treated cells (lane 2); PMA-treated cells (lane 3); sodium arsenite-treated cells (lane 4); dibutyl cAMP-treated cells (lane 5).



To rule out that PMA exposure, like heat shock or sodium arsenite treatment, simply was initiating a classical stress response and an accompanying stress-induced increase in hsp 28 phosphorylation, HL-60 cells were examined for possible changes in the expression of the different stress proteins following their exposure to PMA. In contrast to the increased rates of synthesis of hsp 90, hsp 73, hsp 72, and hsp 28 in cells placed under stress via exposure to sodium arsenite, no obvious changes in the overall expression of the stress proteins were observed in the PMA-treated HL-60 cells during their first 12 h of commitment toward the macrophage-like phenotype (Fig. 2).


Figure 2: PMA stimulation of HL-60 cells does not result in an induction of the stress response. Undifferentiated HL-60 cells were treated with either sodium arsenite (150 µM) or PMA (100 ng/ml) for 90 min. The culture medium was then removed and the cells labeled with [^3H]leucine for 14 h. As a control, untreated cells were labeled in a similar manner. The labeled cells were harvested in LSB, and an equal amount of radiolabeled material (as determined by trichloroacetic acid precipitation) was analyzed by two-dimensional gel electrophoresis. Shown are the fluorographs of the gels. Panel A, control, untreated cells. Panel B, arsenite-treated cells. Panel C, PMA-treated cells. The arrow near the bottom of the gels indicates the position of the of hsp 28 (seen only in the arsenite-treated cells, panel B). The positions of hsp 73 (a), hsp 72 (b), hsp 90 (c), and actin (ac) are indicated.



Complete differentiation of HL-60 cells to the macrophage-like phenotype requires approximately 48-72 h following their initial exposure to the differentiation agent, PMA. Moreover, in the case of PMA, once the cells have become committed to the differentiation process (within a few hours), the PMA can be removed from the cells without affecting the overall differentiation process (reviewed in (42) ). We examined next whether changes in the overall levels of hsp 28 might accompany the transition of the cells into their terminally differentiated phenotype. HL-60 cells were treated with PMA for 4 h, the culture medium containing the drug was removed, and the cells further incubated in complete medium lacking PMA for varying periods of time. At different times during the course of the differentiation process, cells were harvested, and the relative levels of hsp 28 were determined via Western blotting (Fig. 3). For each time point, an equal amount of total cellular protein was applied to the gels. After the first 12 h following administration of PMA, no changes in hsp 28 levels were observed. By 24 h, however, the levels of the protein had increased, and by 36 h the cells exhibited a significant increased accumulation of hsp 28. By 72 h, a time by which greater than 75% of the cells have terminally differentiated into the macrophage-like phenotype, the cells contained relatively high amounts of hsp 28. Again such an increase in the amounts of hsp 28 did not appear to result from the cells having undergone a stress-like response. For example, we did not observe any increases in the expression or accumulation of the other most highly stress-inducible protein, hsp 72, during the course of the PMA-induced differentiation (data not shown but see Fig. 5).


Figure 3: The levels of hsp 28 gradually increase over the course of PMA-induced HL-60 cell differentiation. Undifferentiated HL-60 cells were treated with PMA (100 ng/ml) for 4 h, after which the culture medium was removed, the cells extensively washed with and further incubated in fresh culture medium for various times as indicated below (12-72 h). At the indicated time, the cells were harvested, total protein content determined, and then 20 µg of total protein from each cell lysate was applied to an SDS-gel. Following transfer of the proteins to nitrocellulose, the blots were probed with the mouse monoclonal antibody specific for hsp 28. The primary antibody was visualized by subsequent incubation with horseradish peroxidase-conjugated goat anti-mouse antibody and chemilluminescence. Lane 1, undifferentiated HL-60 cells. Lanes 2-7, hours after PMA treatment began (12 h, 2; 24 h, 3; 36 h, 4; 48 h, 5; 60 h, 6; and 72 h, 7. Shown in lane 8 are HeLa cells grown at 37 °C and in lane 9 HeLa cells subjected to a 43 °C/90 min heat shock treatment and subsequently recovered at 37 °C for 12 h.




Figure 5: Unlike PMA, differentiation of HL-60 cells with either retinoic acid or dimethyl sulfoxide does not result in any changes in the expression of hsp 28. Duplicate plates of HL-60 cells growing at 37 °C were; left untreated (control); exposed to 100 ng/ml of PMA for 4 h, the medium removed, and the cells washed with and further incubated in complete medium lacking PMA for 56 h; exposed to 140 µM dimethyl sulfoxide continuously for 60 h; exposed to 1 mM retinoic acid continuously for 60 h. The cells, treated as described, then were either maintained at 37 °C for an additional 12 h or were subjected to a 43 °C/45-min heat shock treatment and then returned to 37 °C for an additional 12 h. Cells were harvested in LSB, 20 µg of total protein from each cell lysate was applied to an SDS gel, the proteins separated, and then transferred to nitrocellulose. Simultaneous Western blot analyses was performed using monoclonal antibodies specific for hsp 28 and hsp 72. Visualization of the primary antibodies was done by incubation with horseradish peroxidase-conjugated goat anti-mouse antibodies and chemilluminescence. Panel A shows the results using the hsp 72 antibody and panel B the results using the hsp 28 antibody. Lane 1, PMA-differentiated cells incubated at 37 °C; lane 2, Me(2)SO-differentiated cells incubated at 37 °C; lane 3, RA-differentiated incubated at 37 °C; lane 4, undifferentiated cells heat shock-treated; lane 5, PMA-differentiated cells heat shock-treated; lane 6, Me(2)SO-differentiated cells heat shock-treated; lane 7, RA-differentiated cells heat shock-treated.



Having shown that hsp 28 levels increase in the HL-60 cells following their differentiation into a macrophage-like phenotype, we examined whether the protein, observed to exhibit rapid increased phosphorylation immediately following PMA addition (Fig. 1), might still be phosphorylated 72 h after exposure of the cells to PMA. In addition, we wondered whether the fully differentiated cells, like the undifferentiated cells, would still be able to phosphorylate hsp 28 in response to either metabolic stress or the readdition of PMA. HL-60 differentiation toward the macrophage-like phenotype was initiated by the addition of PMA. 4 h later the medium containing the drug was removed, the cells washed with and further incubated in complete medium (in the absence of PMA) for an additional 68 h to allow for complete differentiation. These now differentiated HL-60 cells, along with HeLa cells (as a control), were then labeled with [P]orthophosphate for 1 h and the pattern of phosphoproteins analyzed by two-dimensional gel electrophoresis. In addition both the PMA-differentiated HL-60 cells and HeLa cells were examined for their pattern of hsp 28 phosphorylation following heat shock treatment, exposure to sodium arsenite, or re-exposure to PMA (Fig. 4). In contrast to the 37 °C HeLa cells (Fig. 4E), the PMA-differentiated HL-60 cells incubated at 37 °C exhibited basal phosphorylation of hsp 28, as evidenced by the single phosphorylated isoform (Fig. 4A). Following heat shock treatment, two phosphorylated isoforms of hsp 28 were readily apparent in the HeLa cells (Fig. 4F). In the differentiated HL-60 cells subjected to the heat shock treatment, the relative intensity of the phosphorylated isoform seen in the 37 °C cells increased, along with the appearance of a minor amount of a second isoform (Fig. 4B). Following sodium arsenite treatment, the three previously described hsp 28-phosphorylated isoforms (26, 30) were apparent in both cell types (Fig. 4, C and G). Finally, and as shown previously(26) , PMA treatment resulted in an obvious increased phosphorylation of hsp 28 in the HeLa cells (Fig. 4H). Once differentiated by exposure to PMA 68 h earlier, the HL-60 cells now no longer displayed a significant increase in the phosphorylation of hsp 28 in response to the readdition of PMA (Fig. 4D).


Figure 4: Comparison of hsp 28 phosphorylated isoforms in Hela and PMA-differentiated HL-60 cells subjected to heat shock, sodium arsenite, or PMA treatment. HeLa cells and PMA-differentiated HL-60 cells (72 h after exposure to PMA) were labeled with [P]H(3)PO(4) for 1 h, and the cells, while in the presence of the radiolabel were then: maintained at 37 °C (control); given a 44 °C heat shock treatment; exposed to 200 uM sodium arsenite; treated with 200 ng/ml PMA. After 30 min, the cells were harvested in LSB and an equal percentage of the cell lysates were analyzed by two-dimensional gel electrophoresis. Shown only are those portions of the autoradiograms revealing the position of the hsp 28 isoforms (acidic end is at the left of each panel). In panels A-D are shown the PMA-differentiated HL-60 cells and in panels E-H the HeLa cells. Panels A and E, untreated, control cells; panels B and F, heat shock-treated cells; panels C and G, arsenite-treated cells; panels D and H, PMA-treated cells. The arrows indicate the positions of the various isoforms of hsp 28.



Because PMA induced differentiation of HL-60 cells toward a macrophage-like phenotype resulted in changes in both the expression and phosphorylation of hsp 28, we investigated whether similar changes might accompany the differentiation of the cells toward a granulocyte-like phenotype. Previous studies have shown that both dimethyl sulfoxide (Me(2)SO) and retinoic acid treatment of HL-60 cells results in their differentiation into a granulocyte-like phenotype (again see (42) for review). In contrast to PMA treatment, we saw no evidence of any increase in the levels of hsp 28 accumulating in the HL-60 cells treated with either Me(2)SO or retinoic acid for 60 h, a period of time sufficient for their almost complete differentiation into the granulocyte-like phenotype (Fig. 5B, lanes 2 and 3, respectively). However, both the Me(2)SO and retinoic acid-differentiated cells were clearly capable of expressing hsp 28 as well as the stress-inducible hsp 72 after heat shock treatment (Fig. 5, A and B, lanes 6 and 7, respectively). With regard to possible changes in the phosphorylation state of hsp 28, HL-60 cells continuously exposed to Me(2)SO for 72 h to elicit the granulocyte phenotype were incubated with [P]orthophosphate. The cells were maintained at 37 °C or subjected to the different agents/treatments (e.g. heat shock, arsenite, the calcium ionophore A23187, cAMP, and lysophosphatidic acid) known to result in the rapid increased phosphorylation of hsp 28 in other cell types (as well as in the undifferentiated HL-60 cells). Analysis of the cell lysates by SDS-PAGE revealed changes in the patterns of phosphorylated proteins following these different treatments (data not shown). However, via immunoprecipitation analyses we could not detect any phosphorylated hsp 28, regardless of the treatment used to elicit its phosphorylation (negative data not shown). Thus, differentiation of the HL-60 cells to the granulocyte-like phenotype is not accompanied by either an increase in the levels or phosphorylation state of hsp 28. Moreover, once differentiated toward the granulocyte phenotype the cells no longer are able to mediate phosphorylation of hsp 28 in response to any of the different agents/treatments which normally induce high level phosphorylation of the protein in other cell types or in the undifferentiated HL-60 cells. Although the granulocyte-like cells still appear capable of up-regulating hsp 28 expression, for example following heat shock treatment, they are unable to mediate phosphorylation of the protein.

Another human-derived cell line capable of undergoing in vitro differentiation events is the erythroleukemia K562 cells (43) . These cells in response to added hemin acquire an erythroid-like phenotype (i.e. red blood cell). Following their exposure to PMA however, the cells adopt a megakarocyte-like phenotype (i.e. platelet producing). Consequently, we were curious to know whether changes in hsp 28 expression/phosphorylation might accompany either of these two differentiation processes. Both hemin-differentiated and PMA-differentiated K562 cells were examined for their relative expression of hsp 28 (Fig. 6B). As a positive control, hsp 72 levels were analyzed owing to previous studies showing its increased expression after hemin induced K562 differentiation (44, 45) (Fig. 6A). Little or no expression of hsp 28 was observed in the K562 cells in their resting, undifferentiated state (at the protein concentrations used to program the gels for comparative Western blotting, Fig. 6B, lane 1) Hsp 72 levels, although relatively low, could be detected in the undifferentiated K562 cells (Fig. 6A, lane 1). Both stress proteins were observed to accumulate following heat shock treatment of the undifferentiated cells (Fig. 6, A and B, lane 2). Following exposure to hemin and differentiation toward the erythroid-like phenotype, the levels of both hsp 72 and hsp 28 were found to be increased relative to that observed for the undifferentiated cells (Fig. 6, A and B, lane 3). Heat shock treatment of the hemin-differentiated cells resulted in even higher levels of the two proteins (Fig. 6, A and B, lane 4). In contrast, no expression of either hsp 72 nor hsp 28 was observed in those cells differentiated toward the megakarocyte lineage following PMA exposure (Fig. 6, A and B, lane 5). After heat shock treatment, however, the PMA-differentiated K562 cells did exhibit a typical stress response, increasing their levels of both stress proteins (Fig. 6, A and B, lane 6).


Figure 6: Levels of hsp 28 and hsp 72 increase in hemin-differentiated but not in PMA-differentiated K562 cells. K562 cells were incubated with 30 µM hemin for 24 h to initiate their differentiation into a erythroid-like phenotype, or were incubated with 100 ng/ml of PMA for 72 h to initiate differentiation into a megakaryocyte-like phenotype. Once fully differentiated the cells were either maintained at 37 °C or subjected to a 43 °C/60-min heat shock treatment and subsequent recovery at 37 °C for 8 h. The cells were harvested, total protein content determined, and then 20 µg of total protein applied to a gel. Following separation by SDS-PAGE, the proteins were transferred to nitrocellulose and the relative amounts of both hsp 72 and hsp 28 determined via Western blotting as described earlier. Shown in panel A are the results using the hsp 72 antibody and in panel B the results using the hsp 28 antibody. Lanes 1 and 2, undifferentiated K562 incubated at 37 °C or subjected to heat shock, respectively; Lanes 3 and 4, hemin-differentiated cells incubated at 37 °C or subjected to heat shock, respectively; Lanes 5 and 6, PMA-differentiated cells incubated at 37 °C or subjected to heat shock, respectively.



The ability of the undifferentiated and hemin-differentiated K562 cells to carry out phosphorylation of hsp 28 was examined. The undifferentiated cells and the hemin-differentiated cells were incubated with [P]orthophosphate and then stimulated with the different agents/treatment which, in most cells, results in the rapid increased phosphorylation of the hsp 28 (Fig. 7). The undifferentiated K562 cells maintained at 37 °C, and shown earlier to express little or no hsp 28, contained little if any phosphorylated hsp 28 (Fig. 7A, lane b). Upon stimulation of the undifferentiated K562 cells by heat shock treatment, exposure to sodium arsenite, addition of PMA, or with the protein kinase inhibitor staurosporine, hsp 28 phosphorylation was observed to increase significantly (Fig. 7A, lanes c-f, respectively). In contrast to the undifferentiated K562 cells, the K562 cells exposed to hemin and now which had increased their level of expression of hsp 28 (Fig. 6) also exhibited relatively high level phosphorylation of hsp 28 (Fig. 7B, lane b). Only a slight increase above this basal level of hsp 28 phosphorylation was observed when the hemin-differentiated cells were subjected to heat shock treatment (Fig. 7B, lane c). Interestingly, upon treatment of the hemin-differentiated cells with either sodium arsenite or PMA hsp 28 phosphorylation was further increased (Fig. 7B, lanes d and e, respectively). Finally, the PMA-differentiated K562 cells, which showed little or no expression of hsp 28 (Fig. 6), also exhibited little or no phosphorylation of the protein regardless of the treatment used (negative data not shown). An interesting difference revealed by these studies is the fact that while PMA treatment does stimulate the increased phosphorylation of hsp 28 very early after its addition to the K562 cells (see Fig. 7A, lane e), once the cells have fully differentiated into the megakaryocyte-like phenotype the extent of hsp 28 phosphorylation declines. Indeed, once fully differentiated to the megakaryocyte like phenotype (via PMA) the cells now appear refractory to most of the classical stimuli known to result in increased hsp 28 phosphorylation. In contrast, the hemin-differentiated K562 erythroid-like cells, like the PMA-differentiated HL-60 macrophage-like cells, express hsp 28 at high levels and continue to mediate relatively high phosphorylation of the protein.


Figure 7: Hemin-induced differentiation of K562 cells results in the sustained and high level phosphorylation of hsp 28. Undifferentiated K562 cells along with K562 cells induced to differentiate into an erythroid-like phenotype via a prior 24-h exposure to 30 µM hemin were incubated with [P]H(3)PO(4) in DMEM lacking phosphate for 1 h. While still in the presence of the radiolabel the cells were: left untreated (control); subjected to a 43 °C heat shock treatment; exposed to 200 µM sodium arsenite; treated with 200 ng/ml of PMA; or treated with 50 nM of the protein kinase inhibitor, staurosporine. After 1 h of the different treatments the cells were harvested in LSB, the lysates heated to 95 °C, and an equal percentage of the cell lysate used for immunoprecipitation of hsp 28. Shown is an autoradiograph of the gel. Panel A shows the hsp 28 immunoprecipitation results from the undifferentiated K562 cells while panel B shows the hsp 28 immunoprecipitation results from the hemin-differentiated K562 cells. Lane designations in the two panels are the same. Lane a, immunoprecipitation using preimmune serum from the undifferentiated and hemin-differentiated cells labeled at 37 °C; lanes b-f represent immunoprecipitations using hsp 28 antibody. Lane b, untreated cells; lane c, heat shock-treated cells; lane d, arsenite-treated cells; lane e, PMA-treated cells; lane f, staurosporine-treated cells.



As a final step in our study we examined the relative distribution of both hsp 28 and actin in the PMA-differentiated HL-60 cells. The rationale for such studies was the fact that hsp 28 previously has been suggested to play a role in the regulation of the actin filament cytoskeleton(15, 16, 17) . Moreover, others had reported that accompanying PMA differentiation of HL-60 cells toward the macrophage-like phenotype was an increase in both the substratum adherence properties of the cells and their levels of actin(46) . HL-60 cells, after 72 h of differentiation by PMA exposure, were examined for the simultaneous distribution of both hsp 28 and polymerized F-actin. Hsp 28 distribution was analyzed by indirect immunofluorescence using an antibody specific for the protein while the distribution of F-actin was determined by incubation of the cells with rhodamine-conjugated phalloidin (Fig. 8). Although not shown, the undifferentiated HL-60 cells, when examined by phase-contrast microscopy were small, well rounded cells which adhered very poorly to their substratum. Upon differentiation into the macrophage-like phenotype, the cells now appeared larger, were no longer rounded, appeared to be motile, and were found to adhere rather strongly to their substratum (Fig. 8). In addition the differentiated cells, in most cases, exhibited an apparent co-localization of hsp 28 with a subset of the actin filaments. Such a co-distribution was most obvious in the phase-dense regions near the perimeter of the cells, this likely representing the leading edge of the cells.


Figure 8: A subpopulation of hsp 28 co-localizes with F-actin in PMA-differentiated HL-60 cells. HL-60 cells were induced to differentiate with PMA as described earlier. Cells were differentiated on glass coverslips for 72 h. The cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100 in PBS, and then analyzed for the simultaneous distribution of hsp 28 and F-actin. Hsp 28 locale was determined by incubation with a mouse monoclonal anti-hsp 28 antibody followed by incubation with fluorescein-conjugated goat anti-mouse antibody. F-actin distribution was determined via incubation with rhodamine-conjugated phalloidin. Shown in panel A is the phase-contrast micrograph, in panel B the distribution of hsp 28, and in panel C the distribution of F-actin. The results of two independent experiments (topversusbottom panels) are shown.




DISCUSSION

The studies presented here demonstrate that one of the proteins whose phosphorylation increases during the early stages of HL-60 differentiation toward a macrophage-like phenotype is the low molecular weight heat shock protein, hsp 28. In addition, over the course of their commitment to the macrophage lineage is the gradual increase in the overall levels of hsp 28. Our results are similar to those of Spector et al.(47) , who similarly reported increased phosphorylation and accumulation of the low molecular weight heat shock protein during PMA-induced HL-60 differentiation. On the basis of these observations, we examined whether similar changes in the phosphorylation and expression of hsp 28 might represent a common event accompanying other cell differentiation events in vitro. The impetus for these studies was to establish different in vitro systems where we might study changes in both the expression and phosphorylation of hsp 28. Once established we could further investigate the proposed connection between hsp 28 and components of the cytoskeleton. To our surprise, however, we observed a tremendous variation in the expression and phosphorylation of the protein in several hematopoietic model systems routinely used to study cell differentiation. To summarize, upon PMA stimulation HL-60 cells adopt a macrophage-like phenotype. Commitment toward the macrophage-like phenotype is accompanied by the rapid phosphorylation of hsp 28 and over time, an accumulation of the protein. In contrast, differentiation toward the granulocyte-like phenotype, via exposure of the HL-60 cells to either Me(2)SO or retinoic acid, did not result in any changes in either the expression or phosphorylation of hsp 28. Rather, once differentiated to a granulocyte-like phenotype, the cells now appeared incapable of mediating the phosphorylation of hsp 28 under any of the conditions known to result in the increased phosphorylation of the protein in other cell types, or even in the undifferentiated HL-60 cells. Although apparently unable to mediate hsp 28 phosphorylation, both the Me(2)SO and retinoic acid-differentiated HL-60 cells still were able to increase the synthesis of hsp 28 following heat shock treatment. In the case of the erythroleukemia cell line K562, the levels of both hsp 72 and hsp 28 were found to increase during the course of hemin-induced differentiation. Once fully differentiated, the erythroid-like cells, similar to the PMA-differentiated HL-60 cells (macrophage-like), expressed appreciable amounts of hsp 28. In contrast, addition of PMA, sufficient to elicit the transition of the K562 cells toward the megakaryocyte-like phenotype, did not result in any increased synthesis or phosphorylation of hsp 28. These admittedly somewhat complicated results are summarized in Table 1.



Thus while changes in the expression/phosphorylation of hsp 28 do accompany some in vitro cell differentiation events, the phenomenon is by no means a universal one. In some respects our results appear similar to the lack of consensus regarding hsp 28 levels and the growth status of different cells. In particular, we hypothesized that as the cells exited their cell cycle upon terminal differentiation, there would occur an increase in the expression and overall levels of hsp 28. The premise for such a hypothesis was the fact that in both yeast and certain mammalian cell lines, expression of the low molecular weight hsp increase as the cells reach stationary phase, and in the case of yeast, during sporulation(22, 23, 24) . In addition, others have shown that overexpression of hsp 28 in Ehrlich ascites tumor cells, via transfection of hsp 28 cDNA, results in a slowing down or even complete inhibition of cell growth(48) . There are, however, exceptions to this connection between hsp 28 expression and a decreased rate or inhibition of cell proliferation. For example some cell lines, such as the breast tumor-derived cell line MCF-7, express extremely high levels of hsp 28, yet grow at very high rates in vitro (reviewed in (49) ). In addition, in Drosophila some of the related members of the low molecular weight hsp family are found to accumulate in cells that apparently are undergoing multiple rounds of cell division during the developmental process(20) . Interestingly, a number of drugs that act as teratogens in mammalian systems, and which have been found to inhibit muscle and neuron differentiation in Drosophila, have as a common property their ability to induce the high level expression of one or more of the low molecular weight hsps(50) .

Thus, the possible connection between the low molecular weight hsps and growth control does not appear to be a simple black versus white situation. Clouding the issue as well is the fact that the low molecular weight hsps undergo phosphorylation in response to a bewildering number of agents/treatments which have no common phenotypic end point; e.g. some which stimulate growth while others which appear to inhibit or slow down growth. For example, increased phosphorylation of the low molecular weight hsp in various animal cells is observed in response to various mitogens (e.g. fresh serum or defined growth factors like epidermal growth factor), cytokines (e.g. interleukin 1, tumor necrosis factor), so-called tumor promoters (e.g. PMA, calcium ionophores), and finally different types of metabolic stress (e.g. heat shock treatment, sodium arsenite exposure, 26-30). Not surprisingly, there have been numerous reports of different kinases and phosphatases which function alone or together in mediating the phosphorylation on serine residues of the low molecular weight hsps in different systems. Indeed, two very recent studies have concluded that there may exist at least two distinct pathways by which phosphorylation of hsp 28 is mediated, one more associated with mitogenic activation, while the other perhaps activated by different types of physiologic stress(51, 52) . With regard to the significance of hsp 28 phosphorylation, recent work from two different laboratories have concluded that phosphorylation of hsp 28 may result in the dissociation of the homo-oligomeric state of the protein (normally somewhere between 400-800 kDa) into a stable tetrameric or dimeric form(31, 32) .

Exactly how expression of the low molecular weight hsp is controlled during development and differentiation, either in vivo or in vitro, remains unclear. At least in vitro, the differentiation of human K562 cells toward a red cell-like phenotype following hemin exposure has been shown previously, as well as here, to result in the increased expression of the highly stress-inducible hsp 72 gene(44, 45) . A significant amount of work has shown that the regulation of hsp 72 expression in cells undergoing metabolic stress is controlled by a transcription factor generally referred to as the heat shock transcription factor (HSF-1). Within the last few years additional genes encoding proteins with similar DNA-binding domains as that of HSF have been identified (reviewed in (53) ). Moreover, one of these genes, termed HSF-2, has been shown to be activated during hemin-induced K562 differentiation and is responsible for the aforementioned increase in hsp 72 expression which accompanies the differentiation process(54) . Thus we suspect that the increased levels of hsp 28, which also accompany the hemin-induced differentiation of the K562 cells, is, like hsp 72 expression, mediated by activation of HSF-2. Our observations that hsp 28 expression increases only under certain differentiation stimuli in both the HL-60 cells (i.e. in response to PMA but not Me(2)SO and retinoic acid) and K562 cells (i.e. in response to hemin but not PMA) provides an ideal system to further study possible differences in the activation of HSF-2 during in vitro cell differentiation events.

Our results represent another contribution to study of in vitro cell differentiation and corresponding changes in stress protein expression. A number of studies using different in vitro systems have observed either increased or decreased expression of one or more of the different stress proteins accompanying the differentiation process (reviewed in (55) ). Of potential clinical relevance, Hanash and colleagues (56, 57) have concluded that specific changes in the relative amounts of hsp 28 isoforms (arising from differential phosphorylation) in childhood acute lymphoblastic leukemia carries prognostic significance. One cannot help but wonder whether these latter observations are related to the earlier mentioned studies in Drosophila showing that inappropriate expression and accumulation of one or more of the low molecular weight hsps very often is associated with developmental abnormalities(50) .

A crucial but still unanswered question concerns the biochemical function of the low molecular weight hsps. Once determined, perhaps new insights regarding the reason for the regulated expression/phosphorylation of the low molecular weight hsps during development and differentiation might be realized. Like the well characterized hsp 70 and hsp 60 families of stress proteins, both the low molecular weight hsps and their related counterparts, the alpha-crystallins, have been reported to function as molecular chaperones, facilitating the refolding of denatured polypeptides and/or protecting target proteins from thermal denaturation(11, 12, 13, 14) . However, unlike hsp 70 and hsp 60, a dependence on ATP of such chaperoning activities has not been demonstrated. Perhaps most telling with regards to a possible function were the observations of Miron et al.(15) , reporting that hsp 25 purified from avian sources was capable of inhibiting the growth of actin filaments in vitro. Subsequently, Landry and colleagues (16, 17) reported both changes in the actin cytoskeleton and the acquisition of a thermotolerant-like phenotype in cells overexpressing the mammalian low molecular weight hsp. These observations, as well as previous studies reporting changes in the levels of both actin and actin-associated proteins during the differentiation of myeloid cells in vitro prompted us to examine the locales of hsp 28 and the actin microfilaments. As was shown in the HL-60 cells following their differentiation toward the more motile macrophage-like phenotype, we did observe a portion of hsp 28 to localize in close proximity to the actin filaments, especially those filaments present within the leading edge of the cell. Whether hsp 28, through phosphorylation/dephosphorylation events triggered by various external stimuli, somehow controls changes in the organization of the actin filament network in the macrophage-like cells is currently under study.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM33551 and American Cancer Society Award CB-91A (to W. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by National Institutes of Health Fellowship F32 GM 15526.

To whom correspondence should be addressed: University of California, San Francisco Box 0854, San Francisco, CA 94143. Tel.: 415-476-8546; Fax: 415-206-4123.

(^1)
The abbreviations used are: hsp, heat shock protein; PMA, phorbol ester myristate; Me(2)SO, dimethyl sulfoxide; RA, retinoic acid; PBS, phosphate-buffered saline; LSB, Laemmli sample buffer; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis.

(^2)
W. Welch, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. R. J. King for his generous gift of the monoclonal antibody to hsp 28. In addition we thank members of the Welch laboratory for helpful discussions of the work.


REFERENCES

  1. Morimoto, R., Tissieres, A., and Georgopoulos, C. (1990) in Stress Proteins in Biology and Medicine , Cold Spring Harbor Press, Cold Spring Harbor, NY
  2. Georgopoulos, C., and Welch, W. J. (1993) Annu. Rev. Cell Biol. 9, 601-634 [CrossRef]
  3. Ellis, R., and van der Vies, S. M. (1991) Annu. Rev. Biochem. 60, 321-347 [CrossRef][Medline] [Order article via Infotrieve]
  4. Arrigo, A. P., and Welch, W. J. (1987) J. Biol. Chem. 262, 15359-15369 [Abstract/Free Full Text]
  5. Tissieres, A., Mitchell, H. K., and Tracy, U. M. (1979) J. Mol. Biol. 84, 389-398
  6. Corces, V., Holmgreen, R., Freund, R., Morimoto, R., and Meselson, M. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5390-5393 [Abstract]
  7. McAlister, L. V., Strausberg, S., Kaluga, A., and Finkelstein, D. (1979) Curr. Genet. 1, 63-74
  8. Kelley, P. M., and Schlesinger, M. J. (1978) Cell 15, 1277-86 [Medline] [Order article via Infotrieve]
  9. Hickey, E. D., and Weber, L. A. (1982) Biochemistry 21, 1513-1521 [Medline] [Order article via Infotrieve]
  10. Ingolia, T. D., and Craig, E. A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2360-2364 [Abstract]
  11. Jacob, U., Gaestel, M., Engel, K., and Buchner, J. (1993) J. Biol. Chem. 268, 1517-1520 [Abstract/Free Full Text]
  12. Horwitz, J. Proc. Natl. Acad. Sci. U. S. A. 89, 10449-104453
  13. Wang, K., and Spector, D. (1994). J. Biol. Chem. 269, 13601-13608 [Abstract/Free Full Text]
  14. Nichol, I. D., and Quinlan, R. A. (1994) EMBO J. 13, 945-953 [Abstract]
  15. Miron, T., Vancompernolle, K., Vandekerckhove, J., Wilchek, M., and Geiger, B. (1991) J. Cell Biol. 114, 255-261 [Abstract]
  16. Lavoie, J. N., Hickey, E., Weber, L. A., and Landry, J. (1993) J. Biol. Chem. 268, 24210-24214 [Abstract/Free Full Text]
  17. Lavoie, J. N., Gingras-Breton, G., Tanguay, R. M., and Landry, J. (1993) J. Biol. Chem. 268, 3420-3429 [Abstract/Free Full Text]
  18. Sirotkin, K., and Davidson, N. (1982) Dev. Biol. 89, 196-205 [Medline] [Order article via Infotrieve]
  19. Cheney, C. M., and Shearn, A. (1983) Dev. Biol. 95, 325-333 [Medline] [Order article via Infotrieve]
  20. Pauli, D., Tonka, C., Tissieres, A., and Arrigo, A. P. (1990) J. Cell Biol. 111, 817-828 [Abstract]
  21. Gernhold, M., Knauf, U., Gaestel, M., Stahl, J., and Kloetzel, P. M. (1993) Dev. Genet. 14, 103-111 [Medline] [Order article via Infotrieve]
  22. Kurtz, S., and Lindquist, S. (1984) Proc. Natl. Acad. Sci.U. S. A. 81, 7323-7327 [Abstract]
  23. Bielka, H., Benndorf, R., and Jungham, I. (1988) Biomed. Biochim Acta 47, 557-563 [Medline] [Order article via Infotrieve]
  24. Gaestel, M., Gross, B., Benndorf, R., Strauss, M., Schunk, W., Kraft, R., Otto, A., Stahl,. J., Drabseh, H., and Bielka, H. (1989) Eur. J. Biochem. 179, 209-213 [Abstract]
  25. Welch, W. J. (1990) in Stress Proteins in Biology and Medicine ( Morimoto, R., Tissieres, A., and Georgopoulos, C., ed) pp. 223-278, Cold Spring Harbor Press, Cold Spring Harbor, NY
  26. Welch, W. J. (1985) J. Biol. Chem. 260, 3058-3065 [Abstract]
  27. Kaur, P., Welch, W., and Saklatvala, J. (1989) FEBS Lett. 258, 269-273 [CrossRef][Medline] [Order article via Infotrieve]
  28. Arrigo, A. P. (1990) Mol. Cell Biol. 10, 1276-1280 [Medline] [Order article via Infotrieve]
  29. Guy, G. R., Chua, S. P., Wong, N. S., Ng, S. B., and Tan, Y. H. (1991) J. Biol. Chem. 266, 14343-14352 [Abstract/Free Full Text]
  30. Kim, Y. J., Shjuman, J., Sette, M., and Przybyla, A. (1984) Mol. Cell. Biol. 4, 468-474 [Medline] [Order article via Infotrieve]
  31. Kato, K., Hasegawa, K., Goto, S., and Inaguma, Y. (1994) J. Biol. Chem. 269, 11274-11278 [Abstract/Free Full Text]
  32. Benndorf, R., Haye, K., Ryazantsev, S., Wieske, M., Behlke, J., and Lutsch, G. (1994) J. Biol. Chem. 269, 20780-20784 [Abstract/Free Full Text]
  33. Feuerstein, N., and Cooper, H. L. (1983) J. Biol. Chem. 258, 10786-10793 [Abstract/Free Full Text]
  34. Feuerstein, N., and Cooper, H. L. (1984) J. Biol. Chem. 259, 2782-2788 [Abstract/Free Full Text]
  35. Richards, F. M., Watson, A., and Hickman, J. A. (1988) Cancer Res. 48, 6715-6720 [Abstract]
  36. Welch, W. J., and Feramisco, J. R. (1984) J. Biol. Chem. 259, 4501-4513 [Abstract/Free Full Text]
  37. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354 [Abstract]
  38. Arrigo, A. P., and Welch, W. J. (1987) J. Biol. Chem. 262, 15359-15369 [Abstract/Free Full Text]
  39. Coffer, A., Lewis, K., Brockas, A. J., and King, R. J. B. (1985) Cancer Res. 45, 3681-3693
  40. Welch, W. J., and Suhan, J. P. (1986) J. Cell Biol. 103, 2035-2052 [Abstract]
  41. Peterson, G. L. (1983) Methods Enzymol. 91, 369-372
  42. Collins, S. J. (1987) Blood 70, 1233-1244 [Abstract]
  43. Colamonici, O., Trepel, J. B., and Neckers, L. A. (1986) Megakaryocyte Development and Function (Levine, R., Williams, N., Levin, J., and Evatt., B., eds) pp. 187-191, Alan R. Liss, New York
  44. Singh, M. K., and Yu, J. (1984) Nature 309, 631-633 [Medline] [Order article via Infotrieve]
  45. Theodorakis, N. G., Zand, D. J., Kotzbauer, P. T., Williams, G. T., and Morimoto, R. (1989) Mol. Cell Biol. 9, 3166-3173 [Medline] [Order article via Infotrieve]
  46. Meyer, W. H., and Howard, T. H. (1983) Blood 62, 308-314 [Abstract]
  47. Spector, N. L., Ryan, C., Samson, W., Levine, H., Nadler, L. M., and Arrigo, A. P. (1993) J. Cell. Physiol. 156, 619-625
  48. Knauf, U., Bielka, H., and Gaestel, M. (1992) FEBSLett. 309, 297-302 [CrossRef][Medline] [Order article via Infotrieve]
  49. Dunn, D. K., Whelan, R. D. H., Hill, B., and King, R. J. B. (1993) J. Steroid Biochem. Mol. Biol. 46, 469-479 [Medline] [Order article via Infotrieve]
  50. Buzin, C. H., and Bournias-Vardiabasis, N. (1984) Proc. Natl. Acad. Sci.U. S. A. 81, 4075-4079 [Abstract]
  51. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Liamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037 [Medline] [Order article via Infotrieve]
  52. Freshney, N. W., Rawlinson, L., Guesdon, F., Jones, E., Cowley, S., Hsuan, J., and Saklatvala, J. (1994) Cell 78, 1039-1049 [Medline] [Order article via Infotrieve]
  53. Lis, J., and Wu, C. (1993) Cell 74, 1-4 [Medline] [Order article via Infotrieve]
  54. Sistonen, L., Sarge, K. D., Phillips, B., Abravaya, K., and Morimoto, R. I. (1992) Mol. Cell Biol. 12, 4104-4111 [Abstract]
  55. Heikkila, J. J. (1993) Dev. Genet. 14, 87-91 [Medline] [Order article via Infotrieve]
  56. Strahler, J. R., Kuick, R., Eckerskorn, C., Lottspeich, F., Richardson, B. C., Fox, D. A., Stoolman, L. M., Hanson, C. A., Nichols, D., Tueche, H. J., and Hanash S. M. (1990) J. Clin. Invest. 85, 200-207 [Medline] [Order article via Infotrieve]
  57. Strahler, J. R., Kuick, R., and Hanash, S. M. (1991) Biochem. Biophys. Res. Commun. 175, 134-142 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.