(Received for publication, August 28, 1995; and in revised form, October 23, 1995)
From the
Several types of cell exhibit cell surface protein kinase (ecto-PK) activities with Ser/Thr-specificity. Ecto-PK sharing certain characteristics of protein kinase CK2 can be detached from intact cells by interaction with exogenous substrates (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V.(1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4021-4025). However, a detailed molecular analysis of this ecto-PK was hampered by the vanishingly small amounts of labile enzyme protein obtained by substrate-inducible enzyme release. We now describe the stabilization and enrichment of released ecto-PK by precipitation with polyethylene glycol followed by affinity chromatography on heparin-agarose. Ecto-PK is shown to consist of two separate forms released in tandem, ecto-PK I and ecto-PK II. Comparison with cell homogenates as well as cell surface biotinylation experiments excluded contamination with intracellular PK. Purified ecto-PK I and ecto-PK II exhibit respectively selective phosphorylation of CK1- and CK2-specific peptide substrates, a complementary sensitivity to inhibitory agents and a differential use of the cosubstrates ATP and GTP. Ecto-PK I consists of a 40-kDa moiety; the ecto-PK II is an ensemble of three components of 43- and 40-kDa (catalytic subunits) and a noncatalytic 28-kDa subunit. In addition, components of the ecto-PK II react with CK2-specific antibodies. Further, comparative peptide mapping and the results of mass spectrometry in combination with assignment of amino acid sequences confirmed that ecto-PK II is closely related if not identical to the protein kinase CK2. Assays with intact cells that result in the phosphorylation of a variety of endogenous membrane proteins showed that both ecto-PKs participate, and further, certain ecto-PK substrates become preferentially labeled by one or another of the enzymes, whereas others are phosphorylated by both ecto-PK activities.
The activity of protein kinases (PK) ()is well
established as a major mediator by which cells relay important signals
for cell growth, metabolism, and homeostasis. The recognition of its
potential importance for extracellular events, however, is relatively
recent. Cell surface PK (ecto-PK), due to their exposed location, have
a potential for reception and transduction of external stimuli. Using
extracellular ATP, the ecto-PK activities allow phosphorylation of cell
surface proteins and/or soluble external substrate proteins in the
environment of the cells. Ecto-PK of eucaryotic cells has been related
to a number of biological
phenomena(1, 2, 3, 4, 5, 6, 7, 8) ;
also, certain parasitic protozoa seem to use ectophosphorylation for
interaction with host cells(9) . The biological relevance of
ecto-PK and extracellular protein phosphorylation received
complementary support from abundant evidence for the existence and the
biological activity of the cosubstrate ATP external to cells, partly
transduced by specific surface receptors (for a recent review, see (10) ).
Studies in our laboratory and those of others have
shown cyclic nucleotide-independent and cAMP-dependent types of ecto-PK
activities in a wide range of vertebrate cell
types(11, 12, 13, 14, 15) .
A ubiquitous ecto-PK activity, insensitive to cyclic nucleotides or
Ca, enables viable cells to phosphorylate endogenous
membrane proteins and foreign substrates. The enzymes' properties
agree with those known for intracellular protein kinase CK2 insofar as
acidic prototype substrates were phosphorylated with ATP and GTP as the
phosphoryl group donor and the enzymes displayed sensitivity to the
glycosaminoglycan heparin(11, 16) . A unique feature
is that this ecto-PK can be released from the intact cell through
exogenous protein substrate(17) . The inducible discharge of
cell surface PK is dependent on stimulation by exogenous substrate and
occurs instantly, thus differing basically from exocytosis or
spontaneous shedding. A series of specific criteria established by our
early studies have shown that ecto-PK shedding occurs in a selective
manner, including no intracellular
components(11, 12, 17, 18, 19, 20) .
Structural as well as functional characterization of ecto-PK activities and their appropriate substrates are only just beginning. Recently, we succeeded in the isolation and identification of two major ecto-PK substrates on the cell surface, revealing them to be homologous forms of certain nuclear proteins(21) . On the other hand, the only direct approach to isolation of ecto-PK is the technique of substrate-induced release, which yields at best vanishingly small amounts of enzyme protein. Hence investigations of the molecular properties of the ecto-PK itself are difficult unless sufficient amounts of enzyme protein are available.
The present study aimed at the characterization of substrate-detached ecto-PK from intact HeLa cells was made possible by the development of a concentration procedure for a simultaneous storage and accumulation of enzyme protein for this purpose. Comparison of the data with known intracellular PKs established that two related ecto-PK forms exist at the cell surface and were set free in tandem. Knowledge of their characteristics will be advantageous for the future detection of specific ecto-PK substrates and the role of their phosphorylation.
Figure 1:
Affinity chromatography of ecto-PK on
heparin-agarose. Ecto-PK was released from the surface of intact cells
(2 10
) without or with 0.5 mg/ml phosvitin and
precipitated with 10% PEG as described under ``Experimental
Procedures.'' The precipitates were resolubilized in
chromatography buffer including 0.1 M NaCl, and samples were
loaded to a heparin-agarose column (see ``Experimental
Procedures''). After extensive washing with the same buffer, the
column was eluted with a linear gradient of NaCl (0.1-1 M), and fractions of 2 ml were collected. Aliquots of each
fraction were assayed for PK activity with
[
-
P]ATP or
[
-
P]GTP and phosvitin as the substrate as
described under ``Experimental Procedures.'' Shown are PK
activity profiles from cell supernatants obtained in the presence
(
) or the absence (
) of phosvitin. The inset shows
the profile obtained from cell sonicates (obtained from 2
10
cells) that represent intracellular PK activities. It
should be noted that the activity levels here are significantly
higher.
Figure 7:
Labeling of ecto-PK by cell surface
biotinylation. HeLa cultures (6.4 10
cells total)
were surface-labeled with N-hydroxysuccinimide-biotin under
the conditions described under ``Experimental Procedures,''
and ecto-PK was released from biotinylated cells with phosvitin under
the routine conditions described in the legend of Fig. 1. To
obtain intracellular PKs, the cells after ecto-PK release were washed
twice with isotonic buffer, scraped from the bottoms of culture flasks,
and disrupted by sonication (see ``Experimental
Procedures''). Released ecto-PK from cell supernatants and
intracellular PKs from cell sonicates were proceeded through
heparin-agarose chromatography followed by SDS-PAGE and transfer to
PVDF membrane. Biotinylation was detected by the enhanced
chemiluminescence technique with alkaline phosphatase-conjugated
streptavidin (see ``Experimental Procedures''). The labeling
of material separated by heparin-agarose is shown for peak I activities (A) and peak II activities (B). Lanes 1 show
intracellular PKs from cell sonicates; lanes 2 show the
ecto-PKs released from intact cells. The relevant part corresponding to
the location of the catalytic subunits of 40 and 43 kDa is
presented.
Figure 2:
Comparative phosphorylation of specific
peptide substrates by purified ecto-PK and intracellular PK activities.
Phosphorylation assays using the peptide substrates (indicated by
single letter code) were carried out with 1 µM [-
P]ATP for 12 min as described under
``Experimental Procedures.'' Aliqouts (2 µl) of the
radioactive reaction mixture were analyzed by thin layer chromatography
on cellulose plates and high voltage electrophoresis. Radioactivity was
detected by autoradiography. Shown are
P-labeled peptides
phosphorylated by affinity purified ecto-PK I (lanes 1) and
ecto-PK II (lanes 2). Peptide phosphorylation by authentic
protein kinases CK1 (lanes 3) and CK2 (lanes 4)
served for reference. Positions of radioactively labeled peptides and
free [
-
P]ATP are
indicated.
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 µl containing
[-
P]ATP or
[
-
P]GTP (specific activity, 25 GBq/mmol)
and was stopped by the addition of 100 µl of ice-cold 10%
trichloroacetic acid and 15 µl of 0.63% bovine serum albumin for
coprecipitation on ice (15 min). Under these conditions the peptides
under investigation remained soluble, whereas larger proteins were
precipitated and could be removed by centrifugation (14000
g for 10 min). 2-µl aliquots of the radioactive 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/H
O/acetone (8/2/75/15)
according to Angiolillo et al.(25) . Radioactivity was
detected by autoradiography (X-Omat AR film, Kodak) and quantified by
the method of thin layer chromatography linear analysis (TLC from
Berthold).
Autophosphorylation
was alternatively performed by an ``in gel assay'' following
essentially the renaturation method of Geahlen et
al.(27) . Briefly, affinity purified ecto-PK was separated
by SDS-PAGE on a gel matrix that had been prepared with 1 mg/ml
-casein in the polymerization solution. After the run, excess SDS
was washed out from the gel by incubation in 40 mM Hepes
buffer, pH 7.4, for 5 h with five changes of the solution. The gel was
subsequently transferred to reaction buffer consisting of 25 mM Hepes, pH 7.4, 10 mM MnCl
, and 4 nM [
-
P]ATP (specific activity, >185
TBq/mmol) or [
-
P]GTP (of identical specific
radioactivity) and left for 3 h on a gently rocking platform. Excess
radioactivity was then removed from the gel by extensive washing with
40 mM Hepes, pH 7.4/1% sodium pyrophosphate until the washing
solution was practically free of radioactivity. Detection of
phosphorylated components was by autoradiography of dried gels.
Figure 8:
Effects of the PK-inhibitors CK I-7 and
heparin on cell surface phosphorylation. The phosphorylation of cell
surface proteins with optimal HeLa cell cultures in the presence of
0.75 µM [-
P]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 µM CK I-7 (lane 2)
or 3 µg/ml heparin (lane 3). After the phosphorylated
cells were extensively rinsed with buffer containing 1 mM unlabeld ATP, the cells were immediately lysed by with SDS sample
buffer, and total cellular proteins were separated by SDS-PAGE
(8-15% polyacrylamide gradient) and stained with Coomassie Blue (A). The radiolabeled cell surface proteins were visualized by
exposing gels to autoradiography (B). The level of phosphate
incorporation into certain phosphoproteins (pp) was determined
by phosphor imaging (C). The molecular masses of marker
proteins and the locations of certain phosphoproteins (pp) are
indicated for comparison.
For immunodetection, the PVDF membranes were incubated with specific polyclonal antibodies. Primary antibodies were stained by enhanced chemiluminescence (Western Light Detection kit) using alkaline phosphatase-conjugated secondary antibodies and its specific substrate bisodium 3-[4-methoxyspiro{1,2 dioxethan-3, 2`-(5`chloro)tricyclo[3.3.1.1.]-decan}-4-yl)phenylphosphate. Signals were detected by exposure to x-ray films.
To remove the large surplus of phosvitin, heparin-agarose was used.
It has a high affinity for ecto-PK whereas the bulk protein, phosvitin,
does not bind to this matrix to any significant extent. Material from a
routine ecto-PK preparation (10 cells) after
resolubilization of PEG precipitate was subjected to heparin-agarose
column chromatography as described under ``Experimental
Procedures.'' As shown in Fig. 1, the ecto-PK preparation
was separated by a 0.1-1 M NaCl gradient into two PK
activity peaks when assayed with [
-
P]ATP.
The first peak of phosvitin phosphorylating activity (peak I) eluted at
about 0.6 M NaCl; the second activity (peak II) eluted
slightly above 0.8 M NaCl. When the phosvitin phosphorylation
was conducted with [
-
P]GTP instead of
[
-
P]ATP, it was observed 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 enrichment with an approximately 22000-fold purification for
ecto-PK II (Table 1). Separation of the ecto-PK fractions by
SDS-PAGE revealed that both fractions carried several proteins.
Equivalent amounts of cell homogenate (rather than material from
intact cells) complemented with phosvitin under identical conditions
for PEG precipitation and heparin affinity chromatography (see
``Experimental Procedures'') and exhibited kinase activity
profiles with three phosvitin kinase activity peaks (Fig. 1, inset). Besides the activities corresponding to peak I and
peak II, another major peak of phosvitin phosphorylating activity was
eluted ahead of peak I at 0.5 M NaCl. It is important to
note that about 200-fold higher levels of enzyme activity were obtained
with homogenates from a given number of cells than with supernatant
material from the same number of intact cells. Hence the difference
observed between enzyme activity profiles under both conditions
eliminate the possibility of participation by material from damaged
cells to the activity profile of released ecto-PK. This was confirmed
by using cell surface biotinylation for further control of cell surface
origin.
The evaluation of
the ecto-PK properties was extended using inhibitors of casein kinases,
including the isoquinolin derivative CK I-7 for CK1 and heparin for
CK2. As shown in Fig. 3, CK I-7 preferentially inhibits ecto-PK
I activity, and heparin affected ecto-PK II catalyzed phosphorylation.
The 50% inhibition (I) of ecto-PK I peptide
phosphorylation was obtained at 9.5 µM CK I-7, whereas
this inhibition was not reached in the ecto-PK II assay. Conversely,
the I
values for heparin were determined as 0.16 µg/ml
for the ecto-PK II phosphorylation and were indeterminable in the
ecto-PK I assay. These results confirm the identity of ecto-PK.
Figure 3:
Effects of CK I-7 (A) and heparin (B) on the activity of ecto-PK. The conditions of the
phosphorylation reactions with the specific peptides DDDDVASLPGLRRR by
ecto-PK I () and RRRAADSDDDDD by ecto-PK II (
) and the
measurement of peptide phosphorylation by cellulose thin layer
electrophoresis were performed as described in the legend of Fig. 2. Phosphorylation rates were measured in the presence of
CK I-7 or heparin at concentrations given in the graph. The mean values
of four independent experiments are given ±
S.D.
To
address this problem we took advantage of a PK renaturation test on
substrate-containing SDS-polyacrylamide gels (in gel assays) as
described under ``Experimental Procedures.'' The results show
that ecto-PK I fractions (Fig. 4A) in the presence of
[-
P]ATP label a single phosphoprotein of 40
kDa (lane 1), indicating that this band represents the ecto-PK
I activity. In the case of ecto-PK II, the 43- and 40-kDa components
were labeled (lane 2). In contrast, the 28-kDa polypeptide
detected by autophosphorylation assay in solution (above) was not
labeled under these conditions, suggesting either a noncatalytic
subunit or copurified substrate. When the in gel assay was conducted
with [
-
P]GTP, the ecto-PK I sample did not
autophosphorylate (Fig. 4B, lane 1), as
expected for CK1 enzymes. In contrast, the ecto-PK II 43-kDa component
as well as the 40-kDa polypeptide (lane 2) could use GTP and
became autophosphorylated.
Figure 4:
Autophosphorylation of ecto-PK.
Autophosphorylation reactions with 500 µl of the affinity purified
and ultrafiltration concentrated ecto-PK I and ecto-PK II were carried
out by an in gel assay as described under ``Experimental
Procedures.'' Ecto-PK I (lanes 1) and ecto-PK II (lanes 2) were reacted for 3 h at room temperature either with
4 nM [-
P]ATP (A) or
[
-
P]GTP (B) as indicated, and
incorporation of radioactivity was analyzed by autoradiography. The
mobilities of molecular mass markers are indicated. It should be noted
that the radioactive label was determined to be covalently bound to
protein as evaluated by re-electrophoresis.
Further Western immunoblot analysis was
carried out with a collection of CK2 antibodies against the subunit
,
`, and
(Fig. 5). None of the CK2 antibodies
recognized ecto-PK I (lane 1). 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
,
`, and
subunits. The
determination of the heteromeric composition of ecto-PK II remains to
be elucidated. On the other hand, that ecto-PK I proteins failed to be
stained by any of the CK2 antibodies further indicates the separate
nature of the ecto-enzymes under investigation (no antibodies toward
human forms of CK1 are available at this time).
Figure 5:
Western immunoblot analysis of ecto-PKs.
Separation by SDS-PAGE (12% acrylamide) and Western blotting to PVDF
membranes of affinity purified ecto-PK I (lane 1), ecto-PK II (lanes 2-5), and authentic CK2 holoenzyme (lane
6) as the reference were as described under ``Experimental
Procedures.'' The Western blots were probed with monospecific
antisera against each of the CK2 subunits (lane 3),
` (lane 4), and
(lane 5) or with a mixture
of the three antisera (lanes 1, 2, and 6).
Antibody binding was detected by the enhanced chemiluminescence
technique given under ``Experimental Procedures.'' The
mobilities of molecular mass markers are indicated. It should be noted
that the covalent nature of the radioactivity incorporation was
confirmed by re-electrophoresis of radiolabeled protein
bands.
Because N-terminal amino acid sequences from affinity purified ecto-PK I and II blotted to PVDF membranes were not determinable, we attempted to obtain internal peptide sequences. Due to the sequence variability seen among species for these enzymes, a valid identification on this basis can only be made 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. Proteolytic peptides were separated by
reversed-phase HPLC on a C18-column (see ``Experimental
Procedures''). For comparison, recombinant human protein kinase
CK2
subunit was prepared and digested. A total of 17 tryptic
peptides, referred to as #1 to #17, were resolved by the HPLC. Fig. 6shows very similar HPLC peptide profiles of the
subunit of ecto-PK II (A) versus control CK2
(B), suggesting a highly homologous if not identical
composition of the two enzyme forms. The peptides #9-11 and
#14-17 of both the ecto-PK and the CK2
were chosen for
further analysis by mass spectrometry (MALDI-MS; see
``Experimental Procedures''). The molecular masses of the
tryptic peptides were found to be very similar. In addition, the
tryptic peptides 9, 14, and 15-17 could be matched by
computer-assisted analysis with theoretical partial amino acid
sequences derived from human CK2
(Table 2). Using this
combination of MALDI-MS and sequence determination, at least 27% of the
total amino acid sequence of catalytic subunit of the ecto-PK II was
identified.
Figure 6:
HPLC
chromatography of tryptic peptides: comparison of elution profiles of
ecto-PK II with CK2. Ecto-PK II was isolated from 5.6 10
HeLa-cells by affinity chromatography as in Fig. 1. After
separation of the enzyme subunits by SDS-PAGE and Western blotting to a
PVDF-membrane, the 43-kDa catalytic subunit (
30 pmol) was cut out
and treated with trypsin as described under ``Experimental
Procedures.'' Separately, 50 pmol of recombinant CK2 was
transferred to the blot membrane and digested with trypsin. The tryptic
peptides of the two samples were subjected to reversed-phase HPLC on a
C18 column (see ``Experimental Procedures''). Shown are the
HPLC elution profiles of ecto-PK II (A), recombinant CK2 (B), and trypsin solution alone (C); PK-derived
peptides are numbered, and peptides that represent digestion products
of trypsin are marked by an asterisk.
The release of ecto-PK from intact cells by protein kinase substrates such as phosvitin or casein appears to be a common phenomenon(17, 33, 34) . The ecto-PK shedding occurs as a specific and immediate response of intact cells to stimulus by a protein substrate. At present, the mode of membrane anchoring of the ecto-PK or the mechanism underlying the enzyme release are not known. However, previous experiments (33, 35) have ruled out the possibility that phosphatidyl inositol-specific phospholipase C could cleave ecto-PK activity from intact cells, which excludes a glycosyl phosphatidylinositol anchor such as described for some other cell surface-located proteins(36) . An ecto-PK liberation by specific proteolysis is unlikely because several protease inhibitors with different specificities were 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 the specificity of the substrate-dependent ecto-PK shedding and discount the possibility of a contribution by intracellular PK activities from dead or damaged cells(11, 12, 17) . Firstly, the experiments here were carried out with HeLa cells grown in serum-free medium to reduce any unspecific protein load of the cell supernatants, because serum protein components may stick firmly to cell cultures. Secondly, comparative affinity chromatography with cell supernatants from intact cells and material from cell homogenates treated under identical conditions resulted in different activity profiles having significantly different activity levels. Thirdly, specific cell surface biotinylation resulted in the labeling of both ecto-PK forms, although their 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 indirectly by the absence of properties exhibited by the second ecto-PK released from intact cells, ecto-PK II. Confirmation of the classification was obtained by specific phosphorylation of the CK1 peptide substrates, DDDDVASLPGLRRR and RRKDLHDDEEDEAMSITA, and through sensitivity to CK I-7, a specific CK1 inhibitor. That the ecto-PK I-catalyzed phosphorylation reactions were limited to the use of ATP as the cosubstrate agrees with the other properties common for CK1 enzymes and is also in line with authentic CK1 from rat, which served as the control CK1 enzyme in this study.
Protein kinases CK1 have been described as an ubiquitous enzyme
family implicated in the control of cytoplasmic and nuclear processes (38, 39, 40, 41) . Molecular
analysis has shown the existence of related yet distinct mammalian CK1
isoenzymes, ,
,
, and
in rat brain and testis
(ranging in size from 25 to 55 kDa), which most probably represent
separate gene products(42, 43, 44) . Although
certain isoforms appear to have broad substrate specificity, the
possibility of a different subcellular distribution of these enzymes is
not well studied. CK1 forms in yeast 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 were described(47, 48) .
Whether the ecto-PK I (ecto-CK1) represents these or one of the other
CK1 family members will require additional characterization at the
molecular level.
The identification of ecto-PK II as a protein
kinase CK2-like enzyme was proven by the specific phosphorylation of
the CK2 peptides RRREEETEEE and RRRAADSDDDDD, its typical inhibition by
low concentrations of heparin, and its unique ability to use both ATP
and GTP as cosubstrate. This classification was confirmed by further
characterization including (i) enzyme autophosphorylation data that
showed two (43 and 40 kDa) 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
fragmentation of ecto-PK II and authentic human CK2
, and (iv)
mass spectrometry (MALDI-MS) of HPLC-separated tryptic peptides from
ecto-PK II
and CK2
and microsequencing. The results from
comparison with the intracellular CK2 in particular underline 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, (49) ). This key role was recently underlined by
the major finding that dysregulatedly expressed catalytic subunit of
CK2 acts as an oncogene(50) . Two isoforms of CK2 catalytic
subunits, and
`, encoded by two different genes are known to
date(51, 52) . In addition, a processed CK2
pseudogene (53) and an intronless gene that encodes CK2
(54) have been described. In most tissues the catalytic
subunits
and
` combine with a 28-kDa noncatalytic subunit
, a potent modulator of enzyme activity(55, 56) ,
to form the heterotetrameric holoenzymes
,
`
or
`
. The CK2
/
` to
ratios may vary considerably(57, 58) , and CK2
can also bind to nuclear or cytosolic proteins not related to
(59, 60) .
An interesting open question is the mechanism of the transfer of ecto-PK I and ecto-PK II to the cell surface. There are no signal motifs that would indicate a classical secretory pathway through the ER or the Golgi network(47, 61, 62) . A further possibility for cell surface localization would be direct extrusion of the ecto-PKs from cytoplasm to the extracellular space and binding to cell surface components as detected for the basic fibroblast growth factor(63) , interleukin 1(64) , or lectin L-29(65) . Such a mechanism, however, seems unlikely due to the stability of the ecto-PK against extensive cell washes(11, 17) , which is that expected from integral membrane proteins. Finally, translocation to the cell surface might also be mediated by carriers such as polyamines known to bind to CK1 and CK2 for transport from cytosol to nucleus (66, 67) or to other compartments of the cell(68, 69) . Results from others indicated that certain heat shock proteins may act as carriers for some cell surface proteins(70, 71) , and protein kinase CK2-heat shock 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-PKs prepared through the heparin affinity chromatography and also as detected by autophosphorylation. Such proteins could be in close proximity to ecto-PKs and become detached with the ectoenzymes through the induced release as an entity. The idea of such a complex, a kind of ``ectokinaseosome,'' merits further detailed studies.
The knowledge of two cyclic nucleotide-independent ecto-PKs and the availability of the specific inhibitors have allowed us to begin to dissect their role in cell surface protein phosphorylation. It is clear already from the initial studies presented here that both enzymes participate and also interact in the ectophosphorylation as indicated by the reduction of labeling intensities by either inhibitor, CK I-7 or heparin. Some ectoproteins appeared to be substrates for both ecto-PK I and ecto-PK II, because both inhibitors affected phosphorylation, although site and order of these ectosubstrate phosphorylation are not evident. Interestingly, studies in vitro have shown that CK1 and CK2 have some common substrates as pointed out by Tuazon and Traugh (38) . Furthermore, phosphorylation of extracellular physiological substrates by CKs have been described, e.g. fibrin and fibrinogen(73) , vitronectin(33) , lectin L-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 ectoenzymes. Both kinases contribute to ectophosphorylation of specific endogenous membrane proteins. Interestingly, ecto-CK1 and ecto-CK2 are released by stimulation with exogenous substrate in tandem, a fact not easily detectable as long as the ecto-PKs were not separated. The spatial arrangement of the ecto-PKs including their association with other proteins as well as the mechanism of release remains to be determined. In principle, a substrate-inducible ecto-PK shedding, as shown in this study, might represent a mechanism for down-regulation of ecto-PK on the cell surface and, on the other hand, up-regulation of extracellular PK activities.