Induced Release of Cell Surface Protein Kinase Yields CK1- andCK2-like Enzymes in Tandem*
(Received for publication, August 28, 1995, and in revised form, October 23, 1995)
Jochen Walter, Martina Schnolzer‡, Walter Pyerin§, Volker Kinzel, and Dieter Kubler¶
From the Departments of Pathochemistry, ‡Cell Biology, and §Biochemical Cell Physiology, German Cancer ResearchCenter (Deutsches Krebsforschungszentrum), 69120 Heidelberg, Federal Republic of Germany
Several types of cell exhibit cell surface protein kinase(ecto-PK) activities with Ser/Thr-specificity. Ecto-PKsharing certain characteristics of protein kinase CK2can be detached from intact cells by interaction withexogenous substrates (Kubler, D., Pyerin, W., Burow, E.,and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,4021–4025). However, a detailed molecular analysis ofthis ecto-PK was hampered by the vanishingly smallamounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabili-zation and enrichment of released ecto-PK by precipita-tion with polyethylene glycol followed by affinitychromatography on heparin-agarose. Ecto-PK is shownto consist of two separate forms released in tandem,ecto-PK I and ecto-PK II. Comparison with cell homoge-nates as well as cell surface biotinylation experimentsexcluded contamination with intracellular PK. Purifiedecto-PK I and ecto-PK II exhibit respectively selectivephosphorylation of CK1- and CK2-specific peptide sub-strates, a complementary sensitivity to inhibitoryagents and a differential use of the cosubstrates ATPand GTP. Ecto-PK I consists of a 40-kDa moiety; theecto-PK II is an ensemble of three components of 43- and40-kDa (catalytic subunits) and a noncatalytic 28-kDasubunit. In addition, components of the ecto-PK II reactwith CK2-specific antibodies. Further, comparative pep-tide mapping and the results of mass spectrometry incombination with assignment of amino acid sequencesconfirmed that ecto-PK II is closely related if not iden-tical to the protein kinase CK2. Assays with intact cellsthat result in the phosphorylation of a variety of endog-enous membrane proteins showed that both ecto-PKsparticipate, and further, certain ecto-PK substrates be-come preferentially labeled by one or another of theenzymes, whereas others are phosphorylated by bothecto-PK activities.
The activity of protein kinases (PK)1 is well established as amajor mediator by which cells relay important signals for cellgrowth, metabolism, and homeostasis. The recognition of itspotential importance for extracellular events, however, is rel-atively recent. Cell surface PK (ecto-PK), due to their exposed
location, have a potential for reception and transduction ofexternal stimuli. Using extracellular ATP, the ecto-PK activi-ties allow phosphorylation of cell surface proteins and/or solu-ble external substrate proteins in the environment of the cells.Ecto-PK of eucaryotic cells has been related to a number ofbiological phenomena (1–8); also, certain parasitic protozoaseem to use ectophosphorylation for interaction with host cells(9). The biological relevance of ecto-PK and extracellular pro-tein phosphorylation received complementary support fromabundant evidence for the existence and the biological activityof the cosubstrate ATP external to cells, partly transduced byspecific surface receptors (for a recent review, see Ref. 10).Studies in our laboratory and those of others have shown
cyclic nucleotide-independent and cAMP-dependent types ofecto-PK activities in a wide range of vertebrate cell types (11–15). A ubiquitous ecto-PK activity, insensitive to cyclic nucleo-tides or Ca21, enables viable cells to phosphorylate endogenousmembrane proteins and foreign substrates. The enzymes’ prop-erties agree with those known for intracellular protein kinaseCK2 insofar as acidic prototype substrates were phosphoryl-ated with ATP and GTP as the phosphoryl group donor and theenzymes displayed sensitivity to the glycosaminoglycan hepa-rin (11, 16). A unique feature is that this ecto-PK can bereleased from the intact cell through exogenous protein sub-strate (17). The inducible discharge of cell surface PK is de-pendent on stimulation by exogenous substrate and occursinstantly, thus differing basically from exocytosis or spontane-ous shedding. A series of specific criteria established by ourearly studies have shown that ecto-PK shedding occurs in aselective manner, including no intracellular components (11,12, 17–20).Structural as well as functional characterization of ecto-PK
activities and their appropriate substrates are only just begin-ning. Recently, we succeeded in the isolation and identificationof two major ecto-PK substrates on the cell surface, revealingthem to be homologous forms of certain nuclear proteins (21).On the other hand, the only direct approach to isolation ofecto-PK is the technique of substrate-induced release, whichyields at best vanishingly small amounts of enzyme protein.Hence investigations of the molecular properties of the ecto-PKitself are difficult unless sufficient amounts of enzyme proteinare available.The present study aimed at the characterization of sub-
strate-detached ecto-PK from intact HeLa cells was made pos-sible by the development of a concentration procedure for asimultaneous storage and accumulation of enzyme protein forthis purpose. Comparison of the data with known intracellularPKs established that two related ecto-PK forms exist at thecell surface and were set free in tandem. Knowledge of theircharacteristics will be advantageous for the future detectionof specific ecto-PK substrates and the role of theirphosphorylation.
* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.¶ To whom correspondence should be addressed: Dept. of Pathochem-
istry 0210, German Cancer Research Center (DKFZ), 69120 Heidelberg,Federal Republic of Germany.
1 The abbreviations used are: PK, protein kinase(s) (EC 2.7.1.37);ecto-PK, cell surface PK; PAGE, polyacrylamide gel electrophoresis;PEG, polyethylene glycol; PVDF, polyvinylidene difluoride; HPLC, highpressure liquid chromatography; MALDI-MS, matrix-assisted laser de-sorption/ionization-mass spectrometry.
Materials—Peptides specific for protein kinase CK1, (DDDD-VASLPGLRRR) (22), RRKDLHDDEEDEAMSITA) (23), and protein ki-nase CK2, (RRRAADSDDDDD (24) and RRREEETEEE) (23)), wereprepared by the peptide synthesis unit of the DKFZ. Ethidium bromideand the material for enhanced chemiluminiscence (Western-Light-De-tection kit) was obtained from Serva (Heidelberg, FRG). All other re-agents and chemicals were of the highest grade available. Proteinkinase CK1 isolated from rat liver was kindly supplied by the group ofProf. O. Issinger (University of Homburg, FRG).Cell Cultures—Cultures of HeLa monolayer cells were adapted to
and finally cultivated in serum-free HL-1 medium (Ventrex). Briefly,adaption was carried out by a stepwise reduction of calf serum (5, 2.5,1, or 0.5% or no serum) with two passages at each step. Serum-freegrown cells have been kept for an as yet unlimited number of passages(;90). For experiments the cells were plated in 25- or 175-cm2 tissueculture flasks (Falcon) and raised to densities of 4–6 3 104 cells/cm2
(subconfluent cultures). Cell viability was evaluated after exposure ofthe cells to conditions for the substrate-inducible release of ecto-PKactivity by a number of independent criteria as described earlier (17).Cell Sonicates—Cell Sonicates (representing intracellular PK activ-
ities, used for experiments as detailed in Figs. 1 and 7) were obtained byscraping off the cells (106 cells/ml of chilled buffer) from the cultureflask in the identical buffer used for ecto-PK release (see below) includ-ing 0.5 mg/ml phosvitin. The cell suspension was sonicated (Bransoncell disruptor B15; microtip, step 3; 7-s blasts with 10-s intervals) for 2min and subsequently passed through a 0.2-mm sterile filter (Millipore)to clear it of particulate material; the filtrate referred to as cell sonicatewas kept ice-cold until use.Release of Ecto-PK Activity from Intact Cells into the Supernatant—
Release of ecto-PK activity from intact cells into the supernatant wascarried out in the presence of 0.5 mg/ml phosvitin for 10 min at 37 °C asdescribed previously (17). The supernatant was passed through a0.2-mm sterile filter (Sartolab P-20, Sartorius) to remove detached cellsor any other particulate matter. The cleared supernatants were treatedfurther for enrichment and purification as given below. Small aliquotsof this supernatant served to determine PK activity.Precipitation of Ecto-PK by Polyethylene Glycol—Cell-free ecto-PK
preparation was mixed with polyethylene glycol (PEG 6000) at concen-trations indicated in the individual experiment. The solutions werekept on ice for 45 min before the precipitates were collected by centrif-ugation (12000 3 g for 10 min). Supernatants were discarded, and thepelleted material was stored at 280 °C. PK assays (phosvitin phospho-rylation) with material redissolved in P-Mix (see above) showed that PKactivities could be fully preserved as PEG precipitate.Heparin Affinity Chromatography—Heparin affinity chromatogra-
phy was carried out with heparin-agarose (column size, 1 ml; Pharma-cia) using the technique of fast protein liquid chromatography (Phar-macia). Ecto-PK precipitates were solubilized with buffer (50 mM Tris/HCl, pH 7.4, 10 mM magnesium acetate, 2 mM EDTA, 1.5 mM 1,4-dithiothreitol; and 0.2 mM phenylmethylsulfonyl fluoride), and sampleswere loaded (0.7 ml/min) on a column that had been pre-equilibratedwith buffer at 100 mM NaCl. The column was extensively washed withthe same buffer until the effluent was essentially free of protein. Elu-tion was performed with buffer (as above) containing a linear gradientof 0.1–1 M NaCl. Fractions of 2 ml were collected and, after adjustingthe samples to 0.1 M NaCl by dilution, assayed for PK activity as givenbelow. Fractions with the highest kinase activities (see Fig. 2) wereused for further characterization. If necessary, the eluate fractions wereconcentrated in batches of 2 ml by ultrafiltration (Centricon-10;Amicon).Phosphorylation Assays—Phosphorylation of phosvitin by released
ecto-PK and analysis of incorporated radioactivity by liquid scintillationcounting were as described earlier (12). Optionally, the phosphorylationreaction was carried out in the presence of either the protein kinaseCK1 inhibitor CK I-7 (Seigagaku) or the CK2 inhibitor heparin (Riker-Kettelhak) at concentrations given in the particular experiments.Phosphorylation of enzyme-specific peptide subunits (1 mg/1 ml) by
purified ecto-PK samples was carried out for 10 min in a total of 100 mlcontaining [g-32P]ATP or [g-32P]GTP (specific activity, 25 GBq/mmol)and was stopped by the addition of 100 ml of ice-cold 10% trichloroaceticacid and 15 ml of 0.63% bovine serum albumin for coprecipitation on ice(15 min). Under these conditions the peptides under investigation re-mained soluble, whereas larger proteins were precipitated and could beremoved by centrifugation (14000 3 g for 10 min). 2-ml aliquots of theradioactive samples were spotted on cellulose thin layer plates (Merck)and separated by high voltage electrophoresis (500–600 V for 40 min)
using a buffer of acetic acid/formic acid/H2O/acetone (8/2/75/15) accord-ing to Angiolillo et al. (25). Radioactivity was detected by autoradiog-raphy (X-Omat AR film, Kodak) and quantified by the method of thinlayer chromatography linear analysis (TLC from Berthold).Autophosphorylation of Ecto-PK—Heparin affinity purified ecto-PK
fractions (500 ml) were incubated with 2 mM [g-32P]ATP. After theautophosphorylation reaction had proceeded for 30 min at 30 °C, thesample was mixed with 200 ml of 20% trichloroacetic acid and 10 ml of5% sodium desoxycholate after Bensadoun and Weinstein (26). Thesample was precipitated for 45 min on ice before centrifugation (140003 g for 10 min). The pellets were solubilized in SDS-containing samplebuffer and separated by polyacrylamide gel electrophoresis (SDS-PAGE). Radiolabeled proteins were detected by autoradiography ofdried gels.Autophosphorylation was alternatively performed by an “in gel as-
say” following essentially the renaturation method of Geahlen et al.(27). Briefly, affinity purified ecto-PK was separated by SDS-PAGE ona gel matrix that had been prepared with 1 mg/ml a-casein in thepolymerization solution. After the run, excess SDS was washed outfrom the gel by incubation in 40 mM Hepes buffer, pH 7.4, for 5 h withfive changes of the solution. The gel was subsequently transferred toreaction buffer consisting of 25 mM Hepes, pH 7.4, 10 mM MnCl2, and 4nM [g-32P]ATP (specific activity, .185 TBq/mmol) or [g-32P]GTP (ofidentical specific radioactivity) and left for 3 h on a gently rockingplatform. Excess radioactivity was then removed from the gel by exten-sive washing with 40 mMHepes, pH 7.4/1% sodium pyrophosphate untilthe washing solution was practically free of radioactivity. Detection ofphosphorylated components was by autoradiography of dried gels.Cell Surface Phosphorylation—Cell surface phosphorylation by
ecto-PK activity on intact cells and analysis of radioactively phospho-rylated proteins was carried out for 12 min as detailed earlier (11) in thepresence of the PK inhibitors heparin or CK I-7 at concentrations givenin the particular experiment. Radiolabeled proteins were analyzed byautoradiography and phosphor imaging (PhosphorImager from Molec-ular Dynamics).Cell Surface Biotinylation—Cell surface Biotinylation with N-hy-
droxysuccinimide-biotin (Fluka) was done according to Cole et al. (28)by the addition of freshly prepared N-hydroxysuccinimide-biotin solu-tion (10 mg/ml in Me2SO) to final concentrations of 20 mg/ml and 0.2%dimethyl sulfoxide. After 15 min of reaction time, the cell supernatantswere aspirated, and cells were washed twice with iso-osmotic solutioncontaining 1 mM ethanolamine. Biotinylated cells were used for exper-iments as described in Fig. 8. The detection of biotin-labeled proteins onpolyvinylidene difluoride (PVDF) membranes (Immobilon-P from Mil-lipore) was done with alkaline phosphatase-conjugated streptavidin byenhanced chemiluminiscence (see Western immunoblot analysisbelow).Western Immunoblot Analysis—Proteins separated by SDS-PAGE
were electrotransfered at 200 mA for 2 h to PVDF membranes using thesemi-dry system described by Kyhse-Anderson (29). Protein on PVDFmembranes was stained with Ponceau S or Amido Black.For immunodetection, the PVDF membranes were incubated with
specific polyclonal antibodies. Primary antibodies were stained by en-hanced chemiluminescence (Western Light Detection kit) using alka-line phosphatase-conjugated secondary antibodies and its specific sub-strate bisodium 3-[4-methoxyspiro{1,2 dioxethan-3, 29-(59chloro)-tricyclo[3.3.1.1.]-decan}-4-yl)phenylphosphate. Signals were detectedby exposure to x-ray films.Peptide Mapping by Trypsin Digestion—Peptide mapping by trypsin
digestion of proteins blotted to PVDF membranes were done accordingto Fernandez et al. (30). Briefly, membranes were cut into small pieces(1 3 1 mm) and incubated with 100 mM Tris/HCl, pH 8.0/10% acetoni-trile/1% Triton (RTX-100), including 0.1 mg of trypsin (BoehringerMannheim, sequencing grade)/mg of protein for 24 h at 30 °C. Trypticpeptides were desorbed from PVDF membranes with 0.1% trifluoroace-tic acid under sonification, and collected supernatants were stored at220 °C.HPLC Chromatography—Tryptic peptides were loaded onto a re-
versed-phase HPLC column (C18, Aquabore OD-300 618–222; 22 30.21 cm; 7 mm; Applied Biosystems), washed with 0.1% trifluoroaceticacid, and eluted (100 ml/min at 70 bar) with a linear gradient of 0–80%acetonitrile/0.085% trifluoroacetic acid. Peptides were monitored at220 nm.N-terminal Microsequencing—Proteins were separated by SDS-
PAGE and transferred to PVDF membrane. After location by stainingwith Ponceau S, the desired proteins were cut out and after destainingstored at 220 °C until use. Blotted proteins were applied to an auto-mated 477A protein sequencer (Applied Biosystems) and assayed for
N-terminal sequences by Edman degradation.Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry
(MALDI-MS)—Peptides after separation by HPLC were analyzed byMALDI-MS using a time of flight mass analyzer (VISION 2000; Finni-gan MAT) according to Hillenkamp et al. (31). Samples of 0.5 ml to-gether with 0.5 ml of matrix solution (10 mg of 2,5-dihydrobenzic acid/mlof 0.1% trifluoroacetic acid) were applied to a stainless steel probe tip(“target”). After crystallization of the sample, 30–50 single laser shotspectra (nitrogen laser, 337 nm) were averaged, and the data werefurther processed using the supplier’s software package. Peptides wereidentified by computer-assisted analysis using the SWISSPROT se-quence data bank and the special program package HUSAR (developedat the Department of Molecular Biophysics, German Cancer ResearchCenter, Heidelberg).
RESULTS
Purification of Ecto-PK—Intact HeLa cells grown under se-rum-free conditions were incubated with phosvitin (0.5 mg/ml)for release of ecto-PK activity (“substrate inducible shedding”).Substrate-induced release yielded less than 20 ng of ecto-PKenzyme protein/106 cells as estimated on the basis of a purifiedCK2 preparation of known specific activity. In addition,ecto-PK solutions proved to be rather unstable, because thephosvitin phosphorylating activity disappeared within a fewhours (data not shown). To enrich enzyme ecto-PK and pre-serve its enzymatic activity, enzyme protein was precipitatedwith PEG. The kinase activity could be quantitatively precipi-tated together with phosvitin by PEG at concentrations above10% and subsequently recovered with no loss of enzyme activ-ity. PEG-precipitated ecto-PK, even after storage at 280 °C forup to 1 year, could be restored to full activity.To remove the large surplus of phosvitin, heparin-agarose
was used. It has a high affinity for ecto-PK whereas the bulkprotein, phosvitin, does not bind to this matrix to any signifi-cant extent. Material from a routine ecto-PK preparation (108
cells) after resolubilization of PEG precipitate was subjected toheparin-agarose column chromatography as described under“Experimental Procedures.” As shown in Fig. 1, the ecto-PKpreparation was separated by a 0.1–1 M NaCl gradient into two
PK activity peaks when assayed with [g-32P]ATP. The firstpeak of phosvitin phosphorylating activity (peak I) eluted atabout 0.6 M NaCl; the second activity (peak II) eluted slightlyabove 0.8 M NaCl. When the phosvitin phosphorylation wasconducted with [g-32P]GTP instead of [g-32P]ATP, it was ob-served that the peak II fractions utilized this cosubstrate,corresponding to the known capability of CK2 to use GTP.Ecto-PK I underwent an approximately 9000-fold enrichmentwith an approximately 22000-fold purification for ecto-PK II(Table I). Separation of the ecto-PK fractions by SDS-PAGErevealed that both fractions carried several proteins.Equivalent amounts of cell homogenate (rather than mate-
rial from intact cells) complemented with phosvitin under iden-tical conditions for PEG precipitation and heparin affinity chro-matography (see “Experimental Procedures”) and exhibitedkinase activity profiles with three phosvitin kinase activitypeaks (Fig. 1, inset). Besides the activities corresponding topeak I and peak II, another major peak of phosvitin phospho-rylating activity was eluted ahead of peak I at ;0.5 M NaCl. Itis important to note that about 200-fold higher levels of enzymeactivity were obtained with homogenates from a given numberof cells than with supernatant material from the same numberof intact cells. Hence the difference observed between enzymeactivity profiles under both conditions eliminate the possibilityof participation by material from damaged cells to the activityprofile of released ecto-PK. This was confirmed by using cellsurface biotinylation for further control of cell surface origin.Enzymatic Properties of Affinity Purified Ecto-PK—Further
characterization of the affinity purified ecto-PK activities wascarried out with particular peptide substrates specific for CK1(22, 23) and CK2 (23, 24). The CK1 peptide but not the CK2peptide was phosphorylated by the ecto-PK I activity (Fig. 2,lane 1). Ecto-PK II exclusively phosphorylated the CK2-specificpeptide (Fig. 2, lane 2). The same trend was obtained whenusing other specific peptide substrates, the CK1 peptide RRK-DLHDDEEDEAAMSITA and the CK2 peptide RRREEETEEE.This result correlates ecto-PK I activity with CK1 and ecto-PKII activity with CK2, respectively. Control experiments withauthentic intracellular CK1 (from rat) and CK2 (human) con-firmed these relationships (Fig. 2, lanes 3 and 4).The evaluation of the ecto-PK properties was extended using
inhibitors of casein kinases, including the isoquinolin deriva-tive CK I-7 for CK1 and heparin for CK2. As shown in Fig. 3,CK I-7 preferentially inhibits ecto-PK I activity, and heparinaffected ecto-PK II catalyzed phosphorylation. The 50% inhibi-tion (I50) of ecto-PK I peptide phosphorylation was obtained at9.5 mM CK I-7, whereas this inhibition was not reached in theecto-PK II assay. Conversely, the I50 values for heparin weredetermined as 0.16 mg/ml for the ecto-PK II phosphorylationand were indeterminable in the ecto-PK I assay. These resultsconfirm the identity of ecto-PK.Molecular Properties—Autophosphorylation in solution (see
“Experimental Procedures”) with the ecto-PK I preparationrevealed labeling of a 40-kDa polypeptide. However, in thepresence of polylysin, a modulator of protein kinase CK2 activ-ity (32), the labeling of the 40-kDa component was suppressed,whereas two other polypeptides of 20 and 21 kDa becamephosphorylated instead (data not shown). Circumstantiallythis result indicates the presence either of enzyme subunits orof other substrates in the enzyme preparation. In the case ofecto-PK II activity, three proteins of 43, 40, and 28 kDa wereradiolabeled, but phosphorylation of these proteins was abol-ished in the presence of polylysin (not shown). Because of thepresence of more than a single phosphorylated polypeptide inthe ecto-PK preparations, it was imperative to determinewhich protein represented the catalytic portion of the enzyme.
FIG. 1. Affinity chromatography of ecto-PK on heparin-agar-ose. Ecto-PK was released from the surface of intact cells (2 3 108)without or with 0.5 mg/ml phosvitin and precipitated with 10% PEG asdescribed under “Experimental Procedures.” The precipitates were res-olubilized in chromatography buffer including 0.1 M NaCl, and sampleswere loaded to a heparin-agarose column (see “Experimental Proce-dures”). After extensive washing with the same buffer, the column waseluted with a linear gradient of NaCl (0.1–1 M), and fractions of 2 mlwere collected. Aliquots of each fraction were assayed for PK activitywith [g-32P]ATP or [g-32P]GTP and phosvitin as the substrate as de-scribed under “Experimental Procedures.” Shown are PK activity pro-files from cell supernatants obtained in the presence (●) or the absence(E) of phosvitin. The inset shows the profile obtained from cell sonicates(obtained from 2 3 108 cells) that represent intracellular PK activities.It should be noted that the activity levels here are significantly higher.
To address this problem we took advantage of a PK renatur-ation test on substrate-containing SDS-polyacrylamide gels (ingel assays) as described under “Experimental Procedures.” Theresults show that ecto-PK I fractions (Fig. 4A) in the presenceof [g-32P]ATP label a single phosphoprotein of 40 kDa (lane 1),indicating that this band represents the ecto-PK I activity. Inthe case of ecto-PK II, the 43- and 40-kDa components were
labeled (lane 2). In contrast, the 28-kDa polypeptide detectedby autophosphorylation assay in solution (above) was not la-beled under these conditions, suggesting either a noncatalyticsubunit or copurified substrate. When the in gel assay wasconducted with [g-32P]GTP, the ecto-PK I sample did not auto-phosphorylate (Fig. 4B, lane 1), as expected for CK1 enzymes.In contrast, the ecto-PK II 43-kDa component as well as the40-kDa polypeptide (lane 2) could use GTP and becameautophosphorylated.Further Western immunoblot analysis was carried out with
a collection of CK2 antibodies against the subunit a, a9, and b(Fig. 5). None of the CK2 antibodies recognized ecto-PK I (lane1). However, ecto-PK II produced positive signals (lanes 2–5)that, in agreement with the control CK2 holoenzyme (lane 6),showed the 43-, 40-, and 28-kDa proteins to be a, a9, and bsubunits. The determination of the heteromeric composition ofecto-PK II remains to be elucidated. On the other hand, thatecto-PK I proteins failed to be stained by any of the CK2antibodies further indicates the separate nature of the ecto-enzymes under investigation (no antibodies toward humanforms of CK1 are available at this time).Because N-terminal amino acid sequences from affinity pu-
rified ecto-PK I and II blotted to PVDF membranes were notdeterminable, we attempted to obtain internal peptide se-quences. Due to the sequence variability seen among species for
TABLE ISummary of ecto-PK purification through heparin-agarose chromatography
Ecto-PK from a total of 8 3 108 HeLa cells was obtained by phosvitin-induced release followed by precipitation with PEG and heparin-affinitychromatography as described under “Experimental Procedures.” Protein kinase assays were carried out with phosvitin using the conditions givenunder “Phosphorylation Assays.” One enzyme unit (U) has been defined as the transfer of 1 pmol of phosphate/min/100 mg of phosvitin.
Volume Protein Total activity Specific activity Purification factora
ml mg units units/mg fold
Startingmaterial
45 180b 1232 6.8 1
ecto-PK I 4 0.01c 247 24700 9080ecto-PK II 6 0.004c 370 92500 22670
a Calculation was based on the proportion of total activities of ecto-PK I and ecto-PK II after their separation on heparin-agarose (as in thirdcolumn).
b These values mainly represent phosvitin that has been added for the purpose of ecto-PK release.c Protein measurement was done from comparative protein staining on SDS-polyacrylamide gels (Coomassie) or after western blotting to PVDF
membranes (Amido Black) with authentic CK2 as the reference.
FIG. 2. Comparative phosphorylation of specific peptide sub-strates by purified ecto-PK and intracellular PK activities. Phos-phorylation assays using the peptide substrates (indicated by singleletter code) were carried out with 1 mM [g-32P]ATP for 12 min asdescribed under “Experimental Procedures.” Aliqouts (2 ml) of the ra-dioactive reaction mixture were analyzed by thin layer chromatographyon cellulose plates and high voltage electrophoresis. Radioactivity wasdetected by autoradiography. Shown are 32P-labeled peptides phospho-rylated by affinity purified ecto-PK I (lanes 1) and ecto-PK II (lanes 2).Peptide phosphorylation by authentic protein kinases CK1 (lanes 3) andCK2 (lanes 4) served for reference. Positions of radioactively labeledpeptides and free [g-32P]ATP are indicated.
FIG. 3. Effects of CK I-7 (A) and heparin (B) on the activity ofecto-PK. The conditions of the phosphorylation reactions with thespecific peptides DDDDVASLPGLRRR by ecto-PK I (●) andRRRAADSDDDDD by ecto-PK II (E) and the measurement of peptidephosphorylation by cellulose thin layer electrophoresis were performedas described in the legend of Fig. 2. Phosphorylation rates were meas-ured in the presence of CK I-7 or heparin at concentrations given in thegraph. The mean values of four independent experiments are given 6S.D.
FIG. 4. Autophosphorylation of ecto-PK. Autophosphorylation re-actions with 500 ml of the affinity purified and ultrafiltration concen-trated ecto-PK I and ecto-PK II were carried out by an in gel assay asdescribed under “Experimental Procedures.” Ecto-PK I (lanes 1) andecto-PK II (lanes 2) were reacted for 3 h at room temperature eitherwith 4 nM [g-32P]ATP (A) or [g-32P]GTP (B) as indicated, and incorpo-ration of radioactivity was analyzed by autoradiography. The mobilitiesof molecular mass markers are indicated. It should be noted that theradioactive label was determined to be covalently bound to protein asevaluated by re-electrophoresis.
these enzymes, a valid identification on this basis can only bemade with reference to a known sample also of human origin;when human CK2 is available, human CK1 is not.For determining internal peptide sequences, ;30 pmol of the
43-kDa subunit of ecto-PK II was digested with trypsin. Pro-teolytic peptides were separated by reversed-phase HPLC on aC18-column (see “Experimental Procedures”). For comparison,recombinant human protein kinase CK2a subunit was pre-pared and digested. A total of 17 tryptic peptides, referred to as#1 to #17, were resolved by the HPLC. Fig. 6 shows very similarHPLC peptide profiles of the a subunit of ecto-PK II (A) versuscontrol CK2a (B), suggesting a highly homologous if not iden-tical composition of the two enzyme forms. The peptides #9–11and #14–17 of both the ecto-PK and the CK2a were chosen forfurther analysis by mass spectrometry (MALDI-MS; see “Ex-perimental Procedures”). The molecular masses of the trypticpeptides were found to be very similar. In addition, the trypticpeptides 9, 14, and 15–17 could be matched by computer-assisted analysis with theoretical partial amino acid sequencesderived from human CK2a (Table II). Using this combination ofMALDI-MS and sequence determination, at least 27% of thetotal amino acid sequence of catalytic subunit of the ecto-PK IIwas identified.Cell Surface Biotinylation Separates Ecto-PK I and Ecto-PK
II from Their Related Intracellular Enzymes—That ecto-PK isreleased from the surface of intact cells and not derived fromintracellular sources was retested by using the method of cellsurface labeling with biotin (see “Experimental Procedures”).Biotinylated cells were treated under routine conditions forecto-PK release with phosvitin, and cell supernatants wereharvested. Subsequently the cells after ecto-PK release were
treated by sonification (in the presence of phosvitin) to obtainintracellular kinases. Both types of samples, supernatantsfrom intact cells and the cell sonicates, were then treated inparallel by PEG precipitation and heparin affinity chromatog-raphy. The results (Fig. 7) clearly showed that the ecto-PK I (A,lane 2) was biotinylated, whereas the corresponding materialfrom sonicated cells was not (A, lane 1). Similarly, ecto-PK IIawas biotinylated but not the material of the cell sonicate (B,lanes 1 and 2). These results, together with the previous ones,form compelling evidence for the cell surface origin of thereleased ecto-PKs.Cell Surface Protein Substrates of Ecto-PK I and Ecto-PK
II—The identification of two separate ecto-PK activitiesprompted us to study their substrate specificity further, par-ticularly toward the panel of endogenous membrane proteinsthat become phosphorylated after incubation of intact cellswith extracellular [g-32P]ATP (see the Introduction). Becausethe specific inhibitors of CK activities, CK I-7 and heparin,differentially affect the cell-free forms of ecto-PKs (see Fig. 3)these agents could provide a potential means of partitioningcell surface protein phosphorylation on intact cells as well. Toinvestigate this possibility, intact cells were surface phospho-rylated under the influence of 50 mM CK I-7 and 3 mg ofheparin, under which condition substrate phosphorylation wasfound to be significantly reduced (as in Fig. 3 above). Fig. 8
FIG. 5.Western immunoblot analysis of ecto-PKs. Separation bySDS-PAGE (12% acrylamide) and Western blotting to PVDF mem-branes of affinity purified ecto-PK I (lane 1), ecto-PK II (lanes 2–5), andauthentic CK2 holoenzyme (lane 6) as the reference were as describedunder “Experimental Procedures.” The Western blots were probed withmonospecific antisera against each of the CK2 subunits a (lane 3), a9(lane 4), and b (lane 5) or with a mixture of the three antisera (lanes 1,2, and 6). Antibody binding was detected by the enhanced chemilumi-nescence technique given under “Experimental Procedures.” The mo-bilities of molecular mass markers are indicated. It should be noted thatthe covalent nature of the radioactivity incorporation was confirmed byre-electrophoresis of radiolabeled protein bands.
FIG. 6.HPLC chromatography of tryptic peptides: comparisonof elution profiles of ecto-PK II with CK2. Ecto-PK II was isolatedfrom 5.6 3 109 HeLa-cells by affinity chromatography as in Fig. 1. Afterseparation of the enzyme subunits by SDS-PAGE and Western blottingto a PVDF-membrane, the 43-kDa catalytic subunit (;30 pmol) was cutout and treated with trypsin as described under “Experimental Proce-dures.” Separately, 50 pmol of recombinant CK2 was transferred to theblot membrane and digested with trypsin. The tryptic peptides of thetwo samples were subjected to reversed-phase HPLC on a C18 column(see “Experimental Procedures”). Shown are the HPLC elution profilesof ecto-PK II (A), recombinant CK2 (B), and trypsin solution alone (C);PK-derived peptides are numbered, and peptides that represent diges-tion products of trypsin are marked by an asterisk.
shows the protein pattern obtained by separation of cell pro-teins through SDS-PAGE (A) and the corresponding phospho-protein pattern (B). The phosphorylation of several cell surfaceproteins (pp150, pp120, and pp100) was reduced by both CK I-7(lane 2) and heparin (lane 3). A quantitation of the radioactivelabel by phosphor imaging (C) revealed that CK I-7 reducedphosphorylation of pp150 by about 52%, that of pp120 by 59%,and that of pp100 by 50%, whereas heparin caused 33%, 50%,and 60% reduction, respectively. Phosphorylation of pp50 wasreduced specifically by CK I-7, and that of pp64 was affected byheparin, which suggests that these proteins are substrates ofecto-PK I and ecto-PK II, respectively. In contrast, phosphoryl-ation of other polypeptides such as pp67 or pp48 was onlymarginally affected by the inhibitors. These results collectivelyindicate that ecto-PK I and ecto-PK II not only both participatein the observed ectophosphorylation of cell surface proteins butmay use certain substrates in parallel or sequentially.
DISCUSSION
The release of ecto-PK from intact cells by protein kinasesubstrates such as phosvitin or casein appears to be a commonphenomenon (17, 33, 34). The ecto-PK shedding occurs as aspecific and immediate response of intact cells to stimulus by aprotein substrate. At present, the mode of membrane anchor-ing of the ecto-PK or the mechanism underlying the enzymerelease are not known. However, previous experiments (33, 35)
have ruled out the possibility that phosphatidyl inositol-spe-cific phospholipase C could cleave ecto-PK activity from intactcells, which excludes a glycosyl phosphatidylinositol anchorsuch as described for some other cell surface-located proteins(36). An ecto-PK liberation by specific proteolysis is unlikelybecause several protease inhibitors with different specificitieswere not able to suppress enzyme release (35, 37).The present study adds important criteria that support the
evidence for the cell surface origin of the ecto-PK and thespecificity of the substrate-dependent ecto-PK shedding anddiscount the possibility of a contribution by intracellular PKactivities from dead or damaged cells (11, 12, 17). Firstly, theexperiments here were carried out with HeLa cells grown inserum-free medium to reduce any unspecific protein load of thecell supernatants, because serum protein components maystick firmly to cell cultures. Secondly, comparative affinitychromatography with cell supernatants from intact cells andmaterial from cell homogenates treated under identical condi-tions resulted in different activity profiles having significantlydifferent activity levels. Thirdly, specific cell surface biotinyla-tion resulted in the labeling of both ecto-PK forms, althoughtheir correspondent intracellular PK stayed unlabeled.In the case of ecto-PK I, a relation to protein kinase CK1 was
brought out directly by phosphorylation assays and indirectlyby the absence of properties exhibited by the second ecto-PKreleased from intact cells, ecto-PK II. Confirmation of the clas-sification was obtained by specific phosphorylation of the CK1peptide substrates, DDDDVASLPGLRRR and RRKDLHD-DEEDEAMSITA, and through sensitivity to CK I-7, a specificCK1 inhibitor. That the ecto-PK I-catalyzed phosphorylationreactions were limited to the use of ATP as the cosubstrateagrees with the other properties common for CK1 enzymes andis also in line with authentic CK1 from rat, which served as thecontrol CK1 enzyme in this study.Protein kinases CK1 have been described as an ubiquitous
enzyme family implicated in the control of cytoplasmic andnuclear processes (38–41). Molecular analysis has shown theexistence of related yet distinct mammalian CK1 isoenzymes,a, b, g, and d in rat brain and testis (ranging in size from 25 to55 kDa), which most probably represent separate gene prod-ucts (42–44). Although certain isoforms appear to have broadsubstrate specificity, the possibility of a different subcellulardistribution of these enzymes is not well studied. CK1 forms inyeast carry a prenylation motif (XCC) at their C terminus (45)that might aid their location at the plasma membrane (46).Recently two members of the human CK1 gene family weredescribed (47, 48). Whether the ecto-PK I (ecto-CK1) representsthese or one of the other CK1 family members will requireadditional characterization at the molecular level.
TABLE IIDetermination of peptide masses in tryptic digests of ecto-PK II and CK2a
Tryptic peptides were obtained as described under “Experimental Procedures” and in the text. Masses of the peptides were determined by usingthe MALDI-MS system described under “Experimental Procedures.” Identification of the peptides was done by computer-assisted analysis usingthe SWISSPROT sequence data bank and the program package HUSAR (see “Experimental Procedures”). The calculated monoisotopic masses(MH1) of tryptic products of human CK2a were used as the reference.
Peptide number Observed MH1 of ecto-PKIICK2a
Amino acid sequence (position in CK2a)Observed MH1 Calculated MH1
FIG. 7. Labeling of ecto-PK by cell surface biotinylation. HeLacultures (6.4 3 108 cells total) were surface-labeled with N-hydroxysuc-cinimide-biotin under the conditions described under “ExperimentalProcedures,” and ecto-PK was released from biotinylated cells withphosvitin under the routine conditions described in the legend of Fig. 1.To obtain intracellular PKs, the cells after ecto-PK release were washedtwice with isotonic buffer, scraped from the bottoms of culture flasks,and disrupted by sonication (see “Experimental Procedures”). Releasedecto-PK from cell supernatants and intracellular PKs from cell soni-cates were proceeded through heparin-agarose chromatography fol-lowed by SDS-PAGE and transfer to PVDF membrane. Biotinylationwas detected by the enhanced chemiluminescence technique with alka-line phosphatase-conjugated streptavidin (see “Experimental Proce-dures”). The labeling of material separated by heparin-agarose is shownfor peak I activities (A) and peak II activities (B). Lanes 1 show intra-cellular PKs from cell sonicates; lanes 2 show the ecto-PKs releasedfrom intact cells. The relevant part corresponding to the location of thecatalytic subunits of 40 and 43 kDa is presented.
The identification of ecto-PK II as a protein kinase CK2-likeenzyme was proven by the specific phosphorylation of the CK2peptides RRREEETEEE and RRRAADSDDDDD, its typicalinhibition by low concentrations of heparin, and its uniqueability to use both ATP and GTP as cosubstrate. This classifi-cation was confirmed by further characterization including (i)enzyme autophosphorylation data that showed two (43 and 40kDa) catalytic subunits and a 28-kDa noncatalytic subunit, (ii)immunological reaction to the specific human CK2 antibodies,(iii) tryptic peptide maps that resulted in comparable fragmen-tation of ecto-PK IIa and authentic human CK2a, and (iv) massspectrometry (MALDI-MS) of HPLC-separated tryptic peptidesfrom ecto-PK IIa and CK2a and microsequencing. The resultsfrom comparison with the intracellular CK2 in particular un-derline the high degree of their homology if not identity.Many important physiological substrates of CK2 activities
point to the physiological significance of CK2 in cellular events
(for a recent review see Allende and Allende, Ref. 49). This keyrole was recently underlined by the major finding that dysregu-latedly expressed catalytic subunit of CK2 acts as an oncogene(50). Two isoforms of CK2 catalytic subunits, a and a9, encodedby two different genes are known to date (51, 52). In addition,a processed CK2a pseudogene (53) and an intronless gene thatencodes CK2a (54) have been described. In most tissues thecatalytic subunits a and a9 combine with a 28-kDa noncatalyticsubunit b, a potent modulator of enzyme activity (55, 56), toform the heterotetrameric holoenzymes a2b2, aa9b2 or a29b2.The CK2a/a9 to b ratios may vary considerably (57, 58), andCK2a can also bind to nuclear or cytosolic proteins not relatedto b (59, 60).An interesting open question is the mechanism of the trans-
fer of ecto-PK I and ecto-PK II to the cell surface. There are nosignal motifs that would indicate a classical secretory pathwaythrough the ER or the Golgi network (47, 61, 62). A further
FIG. 8. Effects of the PK-inhibitors CK I-7 and heparin on cell surface phosphorylation. The phosphorylation of cell surface proteinswith optimal HeLa cell cultures in the presence of 0.75 mM [g-32P]ATP was performed as described under “Experimental Procedures.” The reactions(12 min) were carried out in the absence of inhibitors (lane 1) or in presence of 50 mM CK I-7 (lane 2) or 3 mg/ml heparin (lane 3). After thephosphorylated cells were extensively rinsed with buffer containing 1 mM unlabeld ATP, the cells were immediately lysed by with SDS samplebuffer, and total cellular proteins were separated by SDS-PAGE (8–15% polyacrylamide gradient) and stained with Coomassie Blue (A). Theradiolabeled cell surface proteins were visualized by exposing gels to autoradiography (B). The level of phosphate incorporation into certainphosphoproteins (pp) was determined by phosphor imaging (C). The molecular masses of marker proteins and the locations of certain phospho-proteins (pp) are indicated for comparison.
possibility for cell surface localization would be direct extrusionof the ecto-PKs from cytoplasm to the extracellular space andbinding to cell surface components as detected for the basicfibroblast growth factor (63), interleukin 1 (64), or lectin L-29(65). Such a mechanism, however, seems unlikely due to thestability of the ecto-PK against extensive cell washes (11, 17),which is that expected from integral membrane proteins. Fi-nally, translocation to the cell surface might also be mediatedby carriers such as polyamines known to bind to CK1 and CK2for transport from cytosol to nucleus (66, 67) or to other com-partments of the cell (68, 69). Results from others indicatedthat certain heat shock proteins may act as carriers for somecell surface proteins (70, 71), and protein kinase CK2-heatshock protein 90 complexes have been shown to occur (72).In this context, it should be noted that a copurification of
certain yet unidentified proteins occurred with both ecto-PKsprepared through the heparin affinity chromatography andalso as detected by autophosphorylation. Such proteins couldbe in close proximity to ecto-PKs and become detached with theectoenzymes through the induced release as an entity. The ideaof such a complex, a kind of “ectokinaseosome,” merits furtherdetailed studies.The knowledge of two cyclic nucleotide-independent ecto-
PKs and the availability of the specific inhibitors have allowedus to begin to dissect their role in cell surface protein phospho-rylation. It is clear already from the initial studies presentedhere that both enzymes participate and also interact in theectophosphorylation as indicated by the reduction of labelingintensities by either inhibitor, CK I-7 or heparin. Some ecto-proteins appeared to be substrates for both ecto-PK I andecto-PK II, because both inhibitors affected phosphorylation,although site and order of these ectosubstrate phosphorylationare not evident. Interestingly, studies in vitro have shown thatCK1 and CK2 have some common substrates as pointed out byTuazon and Traugh (38). Furthermore, phosphorylation of ex-tracellular physiological substrates by CKs have been de-scribed, e.g. fibrin and fibrinogen (73), vitronectin (33), lectinL-29 (74), or neurochordins (75).Our results clearly indicate that isoforms of protein kinases
CK1 and CK2 are located on the cell surface acting as ectoen-zymes. Both kinases contribute to ectophosphorylation of spe-cific endogenous membrane proteins. Interestingly, ecto-CK1and ecto-CK2 are released by stimulation with exogenous sub-strate in tandem, a fact not easily detectable as long as theecto-PKs were not separated. The spatial arrangement of theecto-PKs including their association with other proteins as wellas the mechanism of release remains to be determined. Inprinciple, a substrate-inducible ecto-PK shedding, as shown inthis study, might represent a mechanism for down-regulationof ecto-PK on the cell surface and, on the other hand, up-regulation of extracellular PK activities.
Acknowledgments—We thank C. Bieler for expert technical assist-ance and H. Horn and J. Richards for the cell culture work. We are alsograteful to Drs. L. Bodenbach and P. Lorenz for providing recombinantenzyme and antibodies, Dr. H. Heid for microsequencing, and Dr. J.Sonka for the suggestion to use PEG for enzyme precipitation. Dr. J.Reed is thanked for discussion and semantic help. We also thank A.Lampe-Gegenheimer for help in manuscript preparation.
REFERENCES
1. Dey, C. S., and Majumder, G. C. (1987) Biochem. Biophys. Res. Commun. 146,422–429
2. Myers, L. K., and Kang, E. S. (1990) Exp. Cell Res. 187, 270–2763. Naik, U. P., Kornecki, E., and Ehrlich, Y. H. (1991) Biochim. Biophys. Acta
1092, 256–2644. Nagashima, K., Nakanishi, S., and Matsuda, Y. (1991) FEBS Lett. 293,
119–1235. Chen, X. Y., and Lo, T. C. Y. (1991) Biochem. J. 279, 475–4826. Kubler, D., Reinhardt, D., Reed, J., Pyerin, W., and Kinzel, V. (1992) Eur. J.
Biochem. 206, 179–186
7. Uriarte, M., Stalmans, W., Hickman, S., and Bollen, M. (1993) Biochem J. 293,93–100
8. Friedberg, I., Belzer, I., Oged-Plesz, O., and Kubler, D. (1995) J. Biol. Chem.270, 20560–20567
9. Hermoso, T, Fishelson, Z., Becker, S. I., Hirschberg, K., and Jaffe, C. L. (1991)EMBO J. 10, 4061–4067
11. Kubler, D., Pyerin, W., and Kinzel, V. (1982) J. Biol. Chem. 257, 322–32912. Kubler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, S., and Kinzel, V. (1989) J.
Biol. Chem. 64, 14549–1455513. Ehrlich, Y. H., Davis, T. B., Bock, E., Kornecki, E., and Lenox, R. H. (1986)
Nature 320, 67–7014. Fantini, J., Muller, J.-M., Abadie, B., El Battari, A., Marvaldi, J., and Tirard,
A. (1987) Eur. J. Cell Biol. 43, 342–34715. Skubitz, K. M., and Goueli, S. A. (1991) Biochem. Biophys. Res. Commun. 174,
49–5516. Pyerin, W., Burow, E., Michaely, K., Kubler, D., and Kinzel, V. (1987) Biol.
Chem. Hoppe-Seyler 368, 215–22717. Kubler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci.
U. S. A. 80, 4021–402518. Kubler, D., Pyerin, W., and Kinzel, V. (1982) Eur. J. Cell Biol. 26, 306–30919. Kinzel, V., Kubler, D., Burow, E., and Pyerin, W. (1986) in Cellular Biology of
Ectoenzymes (Kreutzberg, G. W., Redington, M., and Zimmermann, H., eds)pp. 179–182, Springer-Verlag, Berlin
20. Kubler, D., Pyerin, W., Fehst, M., and Kinzel, V. (1986) in Cellular Biology ofEctoenzymes (Kreutzberg, G. W., Redington, M., and Zimmermann, H.,eds.) pp. 191–204, Springer-Verlag, Berlin
21. Jordan, P., Heid, H., Kinzel, V., and Kubler, D. (1994) Biochemistry 33,14696–14706
22. Flotow, H., and Roach, P. J (1991) J. Biol. Chem. 266, 3724–372723. Marin, O., Meggio, F., and Pinna, L. A. (1994) Biochem. Biophys. Res. Com-
mun. 198, 898–90524. Kuenzel, E. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,
737–74125. Angiolillo, A., Branucci, M., Marsili, V., Panara, F., Micano, A., Amici, D., and
Gianfranceschi, G. L. (1993) Mol. Cell. Biochem. 121, 65–7226. Bensadoun, A., and Weinstein, D. (1976) Anal. Biochem. 70, 241–25027. Geahlen, R. T., Anostario, M., Low, P. S., and Harrison, M. L. (1986) Anal.
Biochem. 153, 151–15828. Cole, S. R., Ashman, L. K., and Ey, P. L. (1987) Mol. Immunol. 24, 699–70529. Kyhse-Anderson, J. (1984) J. Biochem. Biophys. Methods 10, 203–20930. Fernandez, J., Andrews, L., and Mische, S. M. (1994) Anal. Biochem. 218,
112–11731. Hillenkamp, F., Karas, M., Beavis, R. C., and Chait, B. T. (1991) Anal. Chem-
istry 63, 1193–120332. Meggio, F., Brunati, A. M., and Pinna, L. A. (1983) FEBS Lett. 160, 203–20833. Skubitz, K. M., Ehresmann, D. D., and Ducker, T. P. (1991) J. Immunol. 147,
638–65034. Hartmann, M., and Schrader, J. (1992) Biochim. Biophys. Acta 1136, 189–19535. Jordan, P., and Kubler, D. (1992) Biochem. Int. 26, 97–10436. Low, M. G. (1989) FASEB J. 3, 1600–160837. Paas, Y., and Fishelson, Z. (1995) Arch. Biochem. Biophys. 316, 780–78838. Tuazon, P. T., and Traugh, J. A. (1991) Adv. Second Messenger Phosphoprotein
Res. 23, 123–16439. Issinger, O.-G. (1993) Pharmacol. & Ther. 59, 1–3040. Brockman, J. L., Gross, S. D., Sussman, M. R., and Anderson, R. A. (1992)
Proc. Natl. Acad. Sci. U. S. A. 89, 9454–945841. Wang, P., Vancura, A., Mitcheston, T. G. M., and Kuret, J. (1992) Mol. Biol.
Cell 3, 275–28642. Graves, P. R., Haas, D. W., Hagedorn, C. H., DePaoli-Roach, A. A., and Roach,
P. J. (1993) J. Biol. Chem. 268, 6394–640143. Rowles, J., Slaughter, C., Moonaw, C., Hsu, J., and Cobb, M. H. (1991) Proc.
Natl. Acad. Sci. U. S. A. 88, 9548–955244. Zhai, L., Graves, P. R., Longenecker, K. L., DePaoli-Roach, A. A., and Roach,
P. J. (1992) Biochem. Biophys. Res. Commun. 189, 944–94945. Vancura, A., Sessler, A., Leichus, B., and Kuret, J. (1994) J. Biol. Chem. 269,
19271–1927846. Moon, A. L., Janmey, P. A., Louie, K. A., and Drubin, D. G. (1993) J. Cell Biol.
120, 421–43547. Tapia, C., Featherstone, T., Gomez, C., Taillon-Miller, P., Allende, C. C., and
Allende, J. E. (1994) FEBS Lett. 349, 307–31248. Fish, K. J., Cegielska, A., Getman, M. E., Landes, G. M., and Virshup, D. M.
(1995) J. Biol. Chem. 270, 14875–1488349. Allende, J. E., and Allende, C. C. (1995) FASEB J. 9, 313–32350. Seldin, D. C., and Leder, P. (1995) Science 267, 894–89751. Yang-Feng, T. L., Zheng, K., Kopatz, I., Naiman, T., and Canaani, D. (1991)
Nucleic Acids Res. 19, 7125–712952. Wirkner, U., Voss, H., Lichter, P., Ansorge, A., and Pyerin, W. (1994)Genomics
19, 257–26553. Wirkner, U., Voss, H., Lichter, P., Weitz, S., Ansorge, A., and Pyerin, W. (1992)
Biochim. Biophys. Acta 1131, 220–22254. Devilat, I., and Carvallo, P. (1993) FEBS Lett. 316, 114–11855. Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J.,
and Pyerin, W. (1994) Eur. J. Biochem. 220, 263–27356. Meggio, F., Boldyreff, B., Issinger, O.-G., and Pinna, L. A. (1994) Biochemistry
33, 4336–434257. Schmidt-Spaniol, I., Grimm, B., and Issinger, O. G. (1994) Cell. & Mol. Biol.
Res. 39, 761–77258. Mitev, V., Pauloin, A., and Houdebine, L.-M. (1994) J. Neurochem. 63, 717–72659. Stigare, J., Kovacs, J., Buddelmeijer, N., and Egyhazi, E. (1993) FEBS Lett.
314, 327–33060. Molina, E., Plana, M., and Itarte, E. (1991) Biochem. J. 277, 811–818
61. Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227–23362. Meisner, H., Heller-Harrison, R., Buxton, J., and Czech, M. P. (1989) Biochem-
istry 28, 4072–407663. Mignatti, P., Morimoto, T., and Rifkin, D. B. (1992) J. Cell. Physiol. 151, 81–9364. Rubartelli, A. F., Cozzolino, F., Talio, M., and Sitia, R. (1990) EMBO J. 9,
1503–151065. Lindstedt, R., Apodaca, G., Barondes, S. H., Mostov, K. E., and Leffler, H.
(1993) J. Biol. Chem. 268, 11750–1175766. Filhol, O., Cochet, C., and Chambaz, E. M. (1990) Biochem. Biophys. Res.
Commun. 173, 862–87167. Singh, T. J., and Huang, K. (1985) FEBS Lett. 190, 84–8868. Bordin, L., Cattapan, F., Clari, G., Toninello, A., Siliprandi, N., and Moret, V.
(1994) Biochim. Biophys. Acta 1199, 266–270
69. Clari, G., Toninello, A., Bordin, L., Cattapan, F., Piccinelli-Siliprandi, D., andMoret, V. (1994) Biochem. Biophys. Res. Commun. 205, 389–395
70. Winfield, J., Jarjour, W., and Minota, S. (1992) in Heat-Shock Proteins andgamma-delta T Cells (C. F. Brosnan, ed) pp. 47–60, Karger A. G., Basel,Switzerland
71. Heufelder, A. E., Wenzel, B. E., and Bahn, R. S. (1992) J. Clin. Endocrinol. &Metab. 74, 732–736
72. Miyata, Y., and Yahara, I. (1992) J. Biol. Chem. 267, 7042–704773. Sonka, J., Kubler, D., and Kinzel, V. (1989) Biochim. Biophys. Acta 997,
268–27774. Huflejt, M. E., Turck, C. W., Lindstedt, R., Barondes, S. H., and Leffler, H.
(1993) J. Biol. Chem. 268, 26712–2671875. Elizarov, S. M., and Preobrazhensky, A. A. (1993)Mol. Brain Res. 19, 310–312
