From the Adirondack Biomedical Research Institute,
Lake Placid, New York 12946
Protein kinase C (PKC) plays a major role in
regulating cell growth, transformation, and gene expression; however,
identifying phosphorylation events that mediate these responses has
been difficult. We expression-cloned a group of PKC-binding proteins
and identified a high molecular weight, heat-soluble protein as the
major PKC-binding protein in REF52 fibroblasts (Chapline, C., Mousseau,
B., Ramsay, K., Duddy, S., Li, Y., Kiley, S. C., and Jaken, S. (1996) J. Biol. Chem. 271, 6417-6422). In this study,
we demonstrate that this PKC-binding protein, clone 72, is also a PKC
substrate in vitro and in vivo. Using a
combination of phosphopeptide mapping, Edman degradation, and
electrospray mass spectrometry, serine residues 283, 300, 507, and 515 were identified as the major in vitro PKC phosphorylation
sites in clone 72. Phosphorylation state-selective antibodies were
raised against phosphopeptides encompassing each of the four
phosphorylation sites. These antibodies were used to determine that
phorbol esters stimulate phosphorylation of serines 283, 300, 507, and
515 in cultured cells, indicating that clone 72 is directly
phosphorylated by PKC in living cells. Phosphorylated clone 72 preferentially accumulates in membrane protrusions and ruffles,
indicating that PKC activation and clone 72 phosphorylation are
involved in membrane-cytoskeleton remodeling. These data lend further
evidence to the model that PKCs directly interact with, phosphorylate,
and modify the functions of a group of substrate proteins, STICKs
(substrates that interact with
C-kinase).
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INTRODUCTION |
Protein kinase C (PKC)1
is a family of phospholipid-dependent protein kinases
expressed in all cells and tissues (1). PKC activity has been
associated with a variety of growth and pathological defects,
indicating that PKCs are involved in regulating fundamental cellular
processes. PKC is the major cellular receptor for tumor-promoting phorbol esters, and consequently, PKC activation has been linked to
later events in multistage carcinogenesis, i.e. tumor
promotion/progression. Phorbol esters and other PKC activators rapidly
induce a variety of cellular responses including cell shape changes,
cytoskeletal remodeling, decreased cell-cell communication, and
increased exocytosis. The diversity of the observed responses has made
it complicated to focus on particular phosphorylation events that
mediate changes in biological activity. Identifying target proteins and
determining how PKC phosphorylation alters their activities are keys to
understanding the role of PKC in cell regulation.
Several approaches have been used to identify PKC-binding proteins.
PICKs (proteins that interact with
C-kinase) are PKC substrates identified by
yeast two-hybrid screening (2). RACKs (receptors for
activated C-kinase) are
non-substrate proteins that bind to catalytically active PKC. These may
target active PKCs to the vicinity of appropriate substrate proteins
(3). Several years ago, we began using a PKC overlay assay to identify
PKC-interacting proteins (4-6). We found that many of the binding
proteins identified by this method, including MARCKS and the related
protein MacMARCKS, were PKC substrates (5, 7). We therefore named this
group of PKC-binding proteins/substrates STICKs (for
substrates that interact with
C-kinase. In cultured cells, phorbol esters
stimulate 32PO4 incorporation into STICKs (7,
8), indicating that PKC activation increases their phosphorylation.
However, since activating PKC can also lead to increased activity of
other cellular kinases, additional evidence is needed to determine if
the increased phosphorylation is directly mediated by PKC. This is
accomplished by comparing in vitro and in vivo
phosphorylation sites in target proteins. In recent studies, we
determined that two of the PKC-binding proteins we identified,
-adducin and the novel
-adducin (8), are in vivo PKC
substrates by demonstrating that Ser660 is phosphorylated
by PKC in vitro and that phorbol esters stimulate Ser660 phosphorylation in living cells (9). Increased
phosphorylation in transformed renal cells in culture and in renal
tumors was also noted (9, 10). The major PKC-binding protein/substrate detected by overlay assays in normal REF52 fibroblasts (REF A cells) is
a >200-kDa heat-soluble PKC substrate referred to as clone 72 (11) and
also as SSeCKS (12, 13). Clone 72 is also closely related to gravin, an
autoantigen identified in patients with myasthenia gravis (14). Clone
72 protein and message levels are down-modulated in
oncogene-transformed cells, indicating that this protein may play an
important role in normal growth regulation. In fact, reexpressing clone
72 (SSeCKS) in transformed cells has been shown to attenuate growth and
transformation (15). The goal of the present studies was to determine
if clone 72 is an in vivo PKC substrate by comparing
in vitro and in vivo sites of phosphorylation.
Our results indicate that clone 72 is phosphorylated by PKC at multiple
sites in vitro and in vivo. These studies extend our evidence that PKC directly binds to and phosphorylates a group of
target proteins (STICKs). These results contribute to the emerging model that STICKs are primary targets for PKC phosphorylation and that
phosphorylation of STICKs is directly related to the role of PKC in
regulating the complex processes of cell growth, death, and
differentiation.
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MATERIALS AND METHODS |
Preparation of Clone 72 Mutants--
Site-directed mutagenesis
was used to introduce HindIII and Xma sites at
the 5'- and 3'-ends of the binding and phosphorylation domain
encompassing amino acid residues 496-531 (Table I). In this process,
Val497 in the wild type sequence was converted to Leu. This
conservative change did not alter PKC binding, PS binding, or PKC
phosphorylation. The HindIII and Xma sites were
used to insert double-stranded synthetic oligonucleotides that encode
for the deletion mutant sequences 1-4 (Table I). Site-directed
mutagenesis was also used to prepare the single and triple amino acid
changes in mutants 5-7 (Table I). Mutagenesis was done with
double-stranded plasmid DNA using the Chameleon mutagenesis kit
(Stratagene). Mutant proteins were expressed from pTrc (Invitrogen) as
His-tagged fusion proteins. All sequences were verified by sequence
analysis.
Phosphorylation Reactions--
For quantitative measurements,
purified, recombinant proteins and peptides were phosphorylated with
purified recombinant PKC
(PanVera) at 30 °C for 15 min using
standard conditions (4). Phosphorylated proteins were separated on
SDS-polyacrylamide gels and detected by autoradiography. For maximum
phosphorylation needed for identifying phosphorylated residues,
synthetic peptide C34E designed from clone 72 residues
(Lys496-Glu529, with an N-terminal cysteine
added to facilitate coupling) (3.5 µM) was phosphorylated
with PKC
(13.5 nM) for 2-4 h until maximum phosphorylation was reached as monitored by incorporation of
radiolabeled phosphate. The phosphorylated synthetic peptide (6.25 µg) was digested with 0.6 µg of endoproteinase Lys-C overnight. The
digested peptides were analyzed by liquid chromatography electrospray
mass spectrometry (LC-ESMS). Synthetic peptide corresponding to clone 72 residues Arg296-Lys305 (10 µM) was phosphorylated with PKC
(3.0 nM).
The undigested peptide was analyzed by LC-ESMS.
Peptide Synthesis--
Automated FMOC solid phase synthesis was
used to produce PKC phosphorylation substrate peptides R10K and C34E as
well as calibration and tuning reagents for the mass spectrometer
(peptides G9I, K13L, L20R, V26E, E10A, K6R, and bA5K). Peptides G9I
(GRTGRRNSI), K13L (KTREGNVRVSREL), L20R (LKPVKKKKIKREIKILENLR),
V26E (VAMKKIRLEEEGVPSTAIREISLLKE), and C34E
(CKVQSPLKKLFSSSGLKKLSGKKQKGKRGGGGDE) were synthesized at the 250-µmol
scale on HMP (Wang) resin using a Perkin-Elmer Applied Biosystems model
430A Peptide Synthesizer and the Applied Biosystems FastMoc synthesizer
program, cleaved from the solid support with trifluoroacetic acid, and
extracted with acetic acid as described previously (16).
Phosphopeptides E10A (EPQpYEEIPIA-NH2) (where pY represents
phosphorylated tyrosine) and K6R (KRpTIRR-NH2) (where pT
represents phosphorylated threonine) were also prepared on the model
430A instrument (300-µmol scale) using the FastMoc program,
Fmoc-phosphotyrosine (PO3H2)
OH,
Fmoc-phosphothreonine (O-benzyl), and
Fmoc-4-methylbenzylhydrylamine (MBHA) rink amide resin. PKC substrate
peptide R10K (RKKTSFKKSK) (100-µmol scale) and phosphopeptide bA5K
(biotin-ApSSYK) (where pS represents phosphorylated serine) (50-µmol
scale) were synthesized with a Rainin Symphony/Multiplex Peptide
Synthesizer using the synthesizer software (version 3.2) provided by
the instrument manufacturer (Protein Technologies, Inc.) and
Fmoc-phosphoserine (O-benzyl). Following automatic cleavage
from the solid support on the Symphony instrument, peptides were
precipitated from trifluoroacetic acid with ice-cold ether, washed two
or three times with cold ether, and either vacuum-dried or purified by
RP-HPLC. Extended double coupling steps were used for Fmoc-phosphoamino
acids on both instruments; two equimolar additions of the
Fmoc-phosphoamino acid were made approximately 6-12 h apart with a
total coupling reaction time of 18 h. Biotin was coupled with a
single coupling reaction of 12 h. Fmoc-phosphoamino acids,
D-biotin, Wang resins (with or without preloaded Fmoc amino
acids), and Fmoc-MBHA rink amide resin were from AnaSpec. Reagents and
chemicals used with the Symphony instrument were from Rainin, and those
used with the model 430A instrument were from PE Applied Biosystems and Burdick and Jackson. Synthetic peptides used for mass spectrometry tuning and calibration were purified by RP-HPLC using aqueous trifluoroacetic acid/acetonitrile solvents and a 10µ Vydac C18 column
(2.2 × 25 cm). All synthetic peptides were quality
control-analyzed by analytical RP-HPLC and amino acid analysis (17) in
addition to ESMS. Phenylthiocarbamyl amino acid analysis was performed as described previously using an automated analysis system (Applied Biosystems model 420H/130A/920) (18).
Electrospray Mass Spectrometry--
ESMS, LC-ESMS, and low
energy collision tandem MS (MS/MS) were performed with a Perkin-Elmer
Sciex API 300 triple quadrupole mass spectrometer (Thornhill, Canada)
fitted with articulated ion spray plenums and atmospheric pressure
ionization sources. Initial tuning and calibration was with a standard
mixture of polypropylene glycol provided by PE Sciex. Resolution was
adjusted to about 50% valley between adjacent isotope peaks in a
singly charged cluster, allowing singly charged ions to be identified by apparent spacing between peaks and doubly charged ions to be distinguished from those with higher charge states. The nebulization gas for negative ion monitoring was bottled air (Medical Grade USP,
Merriam-Graves, Claremont, NH), and for positive ion monitoring nitrogen was used at 40 p.s.i. Nitrogen was also used as the
curtain gas and was supplied from a Dewar flask (XL-45, Taylor-Wharton) of liquid nitrogen (Merriam-Graves). For infusions in positive ion
mode, spectra were acquired at an orifice potential of 60 V over a scan
range of m/z 300-1500 using 0.25-atomic mass
unit steps and a total scan time of 4.8 s. For negative ion
monitoring, the ion spray needle voltage was operated at
4.0 kV and
the focusing ring at
230 V; for positive ion monitoring, the ion
spray needle voltage was 4.3 kV and the focusing ring was at 300 V. Low
energy collision MS/MS was performed with the RO2 voltage set between
55 and
70 V, nitrogen as the collision gas, and CAD gas settings between 3 and 4, and 50-70 scans were accumulated. Parent ion transmission was maximized by setting Q1 to transmit about a 2-3 m/z window around the selected ion.
LC-ESMS with selective ion monitoring for
PO3
at m/z 79 was performed in negative ion mode at high orifice potential (
190 V)
over a narrow scan range of m/z 78.8-79.2 using
0.1-atomic mass unit steps, a dwell time of 100 ms/step, and a total
scan time of 0.5 s. In the same analysis, full scans in positive
ion mode were acquired at a lower orifice potential (60 V) over the scan range m/z 300-2000 using 0.25-atomic mass
unit steps and a total scan time of 4 s. This was accomplished by
looping the negative ion and positive ion experiments together with
computer-controlled polarity reversal between each scan. The higher
orifice potential in negative ion mode enhances collision-induced
decomposition of phosphopeptides, producing the diagnostic
PO3
fragment ion, thus allowing
selective detection of phosphopeptides (19). The lower orifice
potential used for full scans in positive ion mode allowed acquisition
of mass spectra for all peptides (19). Seven synthetic peptides,
including three phosphopeptides containing phosphotyrosine,
phosphoserine, or phosphothreonine, were used to optimize negative ion
detection sensitivity for the m/z 79 fragment ion
or to serve as internal controls for
PO3
monitoring during LC-ESMS or to
verify correct calibration during LC-ESMS. Synthetic peptides used for
mass spectrometry include G9I (M = 1015.6), K13L (M = 1542.9), L20R (M = 2475.2), V26E (M = 2940.5), and
phosphopeptides E10A (M = 1266.6), K6R (M = 907.5), and b-A5K
(M = 860.4). For LC-ESMS, the HPLC eluant was split, with 22%
going to the mass spectrometer and the remainder collected in 1-min
fractions. RP-HPLC for LC-ESMS was performed at a flow rate of 50 µl/min on a 5µ Vydac C18 microbore column (1 × 250 mm) using
an Applied Biosystems model 120A HPLC system equipped with a 75-µl
dynamic mixer and aqueous acetonitrile/trifluoroacetic acid solvents
(17). Portions of select HPLC fractions were diluted 1:3 in 0.2%
formic acid, 50% methanol and infused at 2 µl/min for MS/MS
analysis.
Clone 72 Antisera--
Four phospho-serine peptides containing
the phosphorylation sites identified in in vitro reactions
were synthesized with N-terminal cysteines added to facilitate coupling
to keyhole limpet hemocyanin and/or solid supports. The peptides were
as follows: Asn276-Lys286 included
phosphoserine 283; Arg296-Lys305 included
phosphoserine 300; Lys503-Lys512 included
phosphoserine 507; and Leu511-Arg523 included
phosphoserine 515. Antisera were raised in rabbits and purified against
the cognate peptides coupled to agarose.
Immunoblotting--
REF52 cells were grown, and cell lysates
were prepared as described previously (7). Aliquots (25-50 µg of
protein) were electrophoresed on SDS-PAGE gels and transferred to
nitrocellulose. Blots were probed with immunopurified antisera
preparations under standard conditions, and reactive bands were
detected with ECL reagents. The phosphorylation state-insensitive
antibody was raised to the recombinant fusion protein containing amino
acid residues 388-790 (clone 72 original) (11).
Immunofluorescence--
REF52 cells were grown on glass
coverslips. Cells were fixed in formaldehyde and permeabilized with
methanol as described previously (20). Cells were incubated with
affinity-purified primary antibodies, washed, and then incubated with
FITC-conjugated species-specific secondary antibodies. Photomicrographs
were taken on a Nikon Optiphot microscope.
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RESULTS |
Mapping PKC Phosphorylation Sites--
In previous studies, we
prepared eight overlapping clone 72 fragments that span the entire open
reading frame. Of the eight recombinant proteins, only fragments 1, 2, and 3 (from the N terminus) were phosphorylated in vitro
with PKC.2 To identify sites
of phosphorylation, the phosphorylated fragments were digested with
proteases, and phosphopeptides were isolated by HPLC and identified by
Edman degradation. Those studies identified three phosphopeptides
containing putative PKC phosphorylation sites,
Thr280-Phe288,
Arg296-Asp308, and
Arg494-Glu530. We have now extended these
studies using mass spectrometry to identify the PKC phosphorylation
sites in these peptides. The synthetic peptide C34E (designed from
residues Lys496-Glu529 with cysteine added)
was phosphorylated with PKC and then digested with endoproteinase
Lys-C. Identified peptides with their corresponding retention times and
masses are summarized in Table I. Several lysyl peptides (representing partially digested fragments) containing a
single serine residue (Ser20 in peptide C34E) corresponding
to serine 515 in clone 72 showed an increased mass of 80 Da, indicative
of phosphate incorporation. Several lysyl peptides containing serine
residues 507-509 also showed an 80-Da increase in mass, indicating
that one of these serine residues is also a target. Observed masses of
lysyl peptides containing serine 500 correlated with calculated masses;
therefore, serine 500 is not a PKC target (Table I).
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Table I
Lysyl peptides identified by LC-ESMS following PKC phosphorylation of
C34E
Synthetic peptide C34E (designed from clone 72 residues; (see
"Materials and Methods") was phosphorylated with PKC, digested with
endoproteinase Lys-C, and analyzed by LC-ESMS (Fig. 6) as described
under "Materials and Methods." Lysyl peptides are listed by
residues, HPLC retention time, calculated and observed masses, and
amino acid sequence. Calculated mass values are based upon monoisotopic
residue weights without phosphate. Underlined sequences are supported
by MS/MS sequence analysis. Potential phosphorylation sites are in
italic type; identified sites are in boldface type.
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To determine which of serines 507-509 were phosphorylated, lysyl
peptide LFSSSGLK (corresponding to residues 505-512 from clone 72) was
further analyzed by low energy collision MS/MS (Fig. 1). The sequence was determined from the
loss in fragment mass starting from the C terminus (y fragments) or N
terminus (b fragments). This analysis confirms that the serine
corresponding to Ser507 in clone 72 is phosphorylated.
Thus, Ser507 and Ser515, but not
Ser500 are in vitro PKC phosphorylation sites in
fragment 3 (Met387-Ile790).

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Fig. 1.
Low energy collision MS/MS analysis of
phosphopeptide L8K. Lysyl peptide LFpSSSGLK identified by LC-ESMS
in Fig. 6 at 34.8 min (MH+ = 918.5) was further analyzed by
MS/MS. Fragment ion nomenclature is as described by Biemann (24).
Mn+ = (M + nH)n+; the asterisks
indicate phosphate losses from the next higher mass (without
H3PO4 (*) and without
HPO3 (**)). pS, phosphorylated serine.
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Phosphorylated residues in the other phosphorylation site found in
fragment 2 (Gln261-Gly532) of clone 72 were
identified by phosphorylating the synthetic peptide corresponding to
residues Arg296-Lys305. Phosphorylation
resulted in an observed mass increase of 80 Da, indicating that only
one of the three serines and threonines is the preferred site (Fig.
2, A and B). Low
energy collision MS/MS analysis showed that phosphorylation occurred at
serine 300 (Fig. 2C).

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Fig. 2.
Electrospray mass spectra of
PKC-phosphorylated peptide R10K and low energy collision MS/MS
analysis. Crude synthetic peptide R10K (RKKTSFKKSK) was
phosphorylated with PKC as described under "Materials and Methods,"
purified by RP-HPLC, and analyzed by ESMS infusion. A, ESMS
spectra of RP-HPLC-purified peptide R10K before phosphorylation
(M2+ = 619.5) (calculated mass = 1236.8; observed
mass = 1236.8). B, ESMS spectra of RP-HPLC-purified
peptide R10K after PKC phosphorylation (M2+ = 659.5)
(observed mass = 1317.0; difference from the observed mass before
phosphorylation = 80.2). C, low energy collision MS/MS
analysis of the doubly charged ion m/z = 659.5 from B above. Nomenclature is as described in the
legend to Fig. 7. pS, phosphorylated serine.
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Comparison of PKC Binding and Phosphorylation Domains--
Several
mutants within the fragment 3 phosphorylation site were constructed to
analyze the structural requirements for PKC binding and phosphorylation
(Table II). Fragment 3 and a subfragment (subfragment 3, Ser370-Glu620) had
quantitatively similar PKC binding activity, indicating that the PKC
binding motif is contained within this region (Fig. 3). Mutating the three tandem serine
residues to alanines (S507-509A) did not influence binding, indicating
that these residues are not critical for PKC binding. Phenylalanine
residues in the MARCKS phosphorylation domain have been shown to be
important for hydrophobic interactions with PS at the membrane
interface (21). Replacing phenylalanine 506 with valine (F506V)
severely attenuated PKC binding and PS binding (Fig. 3). Thus,
interactions of the clone 72 and MARCKS phosphorylation domain peptides
with PS may be somewhat analogous. Structural changes in this motif
were introduced by replacing leucine 511 with proline (L511P).
According to Chou-Fasman and Garnier-Robson rules, this mutation is
predicted to decrease
-structure and increase
-structure. This
mutation also coordinately decreased PKC and PS binding. These results
identify the Lys497-Pro511 region as a major
determinant of phospholipid-dependent PKC binding. Neither of these mutations influenced the net positive charge of this domain, indicating that PKC and PS binding are primarily regulated by hydrophobic interactions and/or secondary structure.
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Table II
Clone 72 mutant constructs
Mutants in the clone 72 phosphorylation site within the
Lys496-Pro531 fragment were prepared and expressed as
His6-tagged fusion proteins.
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Fig. 3.
PKC and PS binding to wild type and mutant
proteins. Wild type and mutant cDNAs were expressed in
bacteria as His6-tagged proteins and purified by nickel
affinity chromatography. Equal amounts of recombinant proteins were
bound to nitrocellulose using a slot blot apparatus. In the clone
72 lane, the nitrocellulose was stained with affinity-purified
clone 72 antibody to compare the amounts of the recombinant proteins.
In the PKC overlay lane, PKC binding was monitored using the
overlay assay (5, 6). In the PS overlay lane,
phosphatidylserine (PS) binding was monitored using
14C-PS. Fragment 3 (72 original), residues
Met387-Ile790; subfragment 3, residues
Ser370-Glu620. F506V, L511P, and S507-509A
are mutants of subfragment 3 shown in Table II.
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As shown in Fig. 4, phosphorylation of
S507-509A relative to the wild type sequence was decreased, which
verifies that these residues are among the PKC targets. Surprisingly,
the F506V and L511P mutants were relatively better PKC substrates than
the wild type protein, despite their decreased PKC and PS binding.
Kinetic analysis of L511P mutant phosphorylation demonstrated that this was due to an increased Vmax with no significant
effect on Km (Fig. 5).
The Vmax value was increased 2.2-fold for the
F506V mutant compared with subfragment 3. Differences in
Km values were <20%. One potential explanation for
this result is that phosphorylatable residues in the wild type sequence
are partially masked, and mutations in this region increase
accessibility to PKC.

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Fig. 4.
PKC phosphorylation of wild type and mutant
proteins. Subfragment 3 and mutational variant proteins (3 µM) were phosphorylated in vitro with PKC in
the absence ( PS) or presence (+PS) of
phosphatidylserine at 30 °C for 15 min. Proteins were resolved by
electrophoresis, and phosphoproteins were detected by autoradiography
(calculated Mr of 50,000). Results presented
were reproduced in two other independent experiments.
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Fig. 5.
Kinetic analysis of clone 72 phosphorylation. Subfragment 3 and mutant F506V (0.13-5
µM) were phosphorylated by PKC in the presence of 1 mM EGTA, 1.2 mM calcium chloride, 20 µg/ml
phosphatidylserine, and 200 nM PDBu at 30 °C for 15 min.
Proteins were resolved by electrophoresis, and phosphoproteins were
detected by autoradiography. Phosphate incorporation was quantitated by
densitometry of the autorad. Data are duplicate measurements.
Lines drawn are best fit determined by linear regression
analysis. Similar results were found in two additional
experiments.
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Clone 72 Is a PKC Substrate in Vivo--
To determine if the PKC
phosphorylation sites identified in in vitro studies were
actual sites in vivo, antisera were raised to
phosphopeptides containing the putative phosphorylation sites Ser300, Ser507, and Ser515. In
addition, an antiserum to Ser283 was prepared, since
preliminary in vitro phosphorylation studies identified
Thr280-Phe289 as a phosphopeptide. Antisera
were affinity-purified against the cognate phosphopeptides.
Specificities of the antibodies were tested by comparing their
reactivities against the phosphorylated and unphosphorylated forms of
the immunizing peptides. As shown in Fig.
6, each of the antibodies preferentially
recognizes the corresponding phosphopeptide compared with the
unphosphorylated peptide. The antibodies did not cross-react with
unphosphorylated recombinant clone 72 proteins. Furthermore, each of
the antibodies specifically recognizes its cognate peptide and does not
cross-react with other Ser(P)-containing peptides. Thus, these
antibodies recognize context-specific Ser(P) and not Ser(P) alone.
These data indicate that these antibodies are phosphorylation
state-selective and can be used to monitor clone 72 phosphorylation at
selected residues.

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Fig. 6.
Characterization of phosphorylation state
selective antisera. Antisera raised to phosphopeptides containing
the PKC phosphorylation sites were affinity-purified against the
cognate phosphopeptides. Purified antibodies (Ab) (1 µg/ml) were reacted with 250 ng of the phosphopeptide or the
unphosphorylated peptide slot blotted onto nitrocellulose. For
phosphoserines 300, 507, and 515, the unphosphorylated peptide was a
corresponding synthetic sequence as indicated. For phosphoserine 283, the unphosphorylated peptide was derived from the phosphopeptide after
exhaustive digestion with alkaline phosphatase.
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To determine if clone 72 is phosphorylated by PKC in vivo,
we prepared extracts from REF52 cells that had been treated with the
PKC activator, PDBu. As shown in Fig. 7,
phorbol esters stimulated phosphorylation at serines 283, 300, 507, and
515. The relative increases in antibody binding were determined by
densitometry of immunoblots and were 634 ± 351, 297 ± 185, 1996 ± 875, and 783 ± 300 (means ± S.D., n = 4)
for phosphoserines 283, 300, 507, and 515, respectively. The correlation between the phosphorylation sites
identified in in vitro and in vivo assays,
indicates that clone 72 is an endogenous PKC substrate.

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Fig. 7.
Clone 72 is phosphorylated by PKC in
vivo. Lysates were prepared from control and PDBu-treated
(100 nM, 10 min) cultures of REF52 cells. Aliquots
(approximately 50 µg of protein) were electrophoresed and blotted to
nitrocellulose. Replicate blots were stained with the indicated
affinity-purified antibodies. The phosphorylation state-insensitive
antibody was raised to recombinant fragment 3 (clone 72 original).
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Clone 72 and Phosphorylated Clone 72 Subcellular Location--
To
gain insight into the function of clone 72 and the effects of PKC
phosphorylation, we compared the localization of clone 72 and
phosphorylated clone 72 by immunofluorescence. Each of the clone 72 antibodies used for immunofluorescence preferentially recognized only
clone 72 on immunoblots of REF52 cell extracts (Fig.
8A), which demonstrates their
suitability for immunolocalization studies. In unstimulated cells,
total clone 72 (monitored with a phosphorylation state nonspecific
antibody) is distributed throughout the cytoplasm and concentrated in
membrane ruffles (Fig. 8B). In contrast, phosphoserine 300 and 515 antibodies did not detect substantial cytoplasmic staining.
With these antibodies, staining was concentrated in membrane
protrusions and ruffles. These data indicate that under physiological
conditions, PKC activation and clone 72 phosphorylation are
preferentially localized to the cell perimeter. The prominent nuclear
staining observed with the phosphoserine 515 antibody is not seen with
other antibodies and appears to be an artifact of this particular
antibody. PDBu caused a redistribution of total clone 72 toward the
perinuclear space but did not substantially influence the staining
intensity. In contrast, PDBu substantially increased the immunostaining
seen with the phosphoserine 300 and 515 antibodies. Staining was
concentrated in the perinuclear region of the cytoplasm but was also
apparent in membrane protrusions and ruffles. PKC isozymes expressed in
these cells (
,
,
, and
) are also found in these locations.
However, the broad distribution patterns of PKCs and clone 72 do not
permit the detailed comparison required to assess colocalization. Other
approaches will be required to determine which PKCs interact with and
phosphorylate clone 72 in vivo.

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Fig. 8.
Immunofluorescence localization of clone 72 and phosphorylated clone 72. A, lysates were
prepared from control and PDBu-treated cultures (200 nM, 10 min). Blots were stained with the phosphorylation
state-insensitive clone 72 antibody (Ab) (Total)
and the phosphorylation state-selective phosphoserine 300 and 515 antibodies (pSer300 and pSer525, respectively).
Full-length, long exposure blots are shown to verify single band
specificity and the suitability of these antibodies for
immunofluorescence studies. B, REF52 cells were grown on
glass coverslips, fixed, and stained as described. Where indicated,
cells were treated with PDBu (200 nM) for 10 min.
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Since PDBu is cell-permeable, it can activate PKCs throughout the cell,
and its actions are not limited to the cell surface. To investigate
physiological PKC activation and clone 72 phosphorylation, cells grown
on coverslips were artificially wounded by scraping the monolayer with
a sharp object. Cells were then stained with antibodies to actin and
clone 72 (Fig. 9). The double staining clearly demonstrates that phosphorylated clone 72 is concentrated in
membrane ruffles and regions in which actin is depolymerized. These
results demonstrate a strong correlation among PKC activation, clone 72 phosphorylation, and dynamic actin remodeling.

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Fig. 9.
Comparison of phosphorylated clone 72 and
actin immunostaining. REF52 cells were grown on glass coverslips
to confluence. The monolayer was artificially wounded by scraping with
a sharp object. After 4-8 h, coverslips were fixed and stained with
mouse monoclonal antibody to -actin and affinity-purified clone 72 rabbit antibodies. After washing, coverslips were stained with
fluorescein- and rhodamine-conjugated species-specific secondary
antibodies to compare actin and phosphorylated clone 72 subcellular
locations.
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DISCUSSION |
PKC phosphorylates a wide variety of proteins in in
vitro reactions, many of which are not relevant physiological
substrates in vivo. The relatively limited number of PKC
phosphorylation events that occur in vivo indicates that PKC
access to substrates is regulated in cells. One mechanism for limiting
PKC phosphorylations to appropriate substrate proteins would be through
direct, high affinity interactions of PKC with its substrates. We have
cloned several PKC-interacting proteins based on their ability to bind PKC in an in vitro assay (4). Binding proteins identified by this method include the PKC substrates MARCKS and the related protein
MacMARCKS (22) and two forms of adducin,
-adducin and the novel
-adducin (8). We named this class of PKC binding proteins/substrates
STICKs (for substrates that
interact with C-kinase. Studying
the effects of PKC phosphorylation on STICK functions provides an
avenue for understanding the mechanisms by which PKC regulates cell
growth, death, and gene expression. In the present study, we
demonstrated that clone 72, the major PKC-binding protein in REF52
cells, is phosphorylated by PKC at the same sites in vitro
and in vivo. In other studies, we found that overexpressing
certain PKCs increases clone 72 phosphorylation, whereas inhibiting PKC
with bisindoylmaleimide I decreases clone 72 phosphorylation.3 We conclude
that PKC is a clone 72 kinase. The selective localization of
phosphorylated clone 72 compared with total clone 72 indicates that
phosphorylation occurs at discrete sites and may be either a
consequence or an effector of localized actin remodeling involved in
formation of membrane protrusions and ruffles.
Clone 72 is related to gravin, which was also recently shown to be a
PKC-binding protein in vitro (14). Gravin is >80%
identical to clone 72 in the PKC binding domains/phosphorylation sites
that we have identified in clone 72. Studies with gravin failed to detect any evidence for an in vivo complex formation between
gravin and PKC (14). However, since gravin is highly homologous to clone 72 in the PKC phosphorylation sites/binding domains, it seems
likely that gravin is also a PKC substrate that associates (at least
transiently) with PKC in vivo. Although a fragment of gravin
was reported to inhibit PKC phosphorylation of an exogenous peptide
substrate, since clone 72 and (by analogy) gravin are PKC substrates,
it seems likely that emphasis should be placed on the role of clone 72 and gravin as PKC substrates rather than inhibitors.
Mutational analysis was used in structure-function studies to define
the PKC binding site in the fragment 3 region of clone 72. We found
that the F506V and L511P mutants had decreased PS and PKC binding
activity yet were improved PKC substrates due to an increase in
Vmax. These results lend support for the model proposed by Ohno et al. (23) to explain why MARCKS is a high affinity (low Km) yet slowly phosphorylated (low
Vmax) PKC substrate. It is possible that high
affinity interactions of PKC with substrates are accomplished at the
expense of the rate of the phosphorylation reaction. For example, the
decreased binding of the F506V and L511P mutants would facilitate
dissociation of product and increase enzyme turnover. Given the high
affinity of PKC for STICKs and the less than optimal phosphorylation
efficiency, it is possible that PKC-STICK interactions are important
for localizing PKC.
In summary, these data provide additional evidence that PKC directly
interacts with and phosphorylates a class of substrate proteins named
STICKs. Thus, STICKs have two important activities: PKC binding
partners and PKC substrates. Phosphorylation of certain STICKs, such as
MARCKS and adducins, alters their functions with regard to association
with cytoskeletal proteins and membranes (20, 21). Thus, STICKs are
also effectors of PKC activation. Additional studies of PKC-STICK
interactions and phosphorylation-induced changes in STICK functions
should provide new insight into the initial events in PKC activation
that contribute to downstream effects on cell growth, transformation,
and gene expression.