(Received for publication, May 3, 1995; and in revised form, July 10, 1995)
From the
The primary structure of rat protein kinase C II was probed
by high pressure liquid chromatography directly coupled to an
electrospray ionization mass spectrometer and by high energy
collision-induced dissociation analysis to identify in vivo phosphorylation sites. The N-terminal methionine was found to be
cleaved post-translationally and replaced with an acetyl group. Four
phosphopeptides were identified. Two peptides,
Thr
-Lys
and
Glu
-Lys
, are phosphorylated at Thr
greater than 90%. Peptide His
-Arg
is
phosphorylated about 75% at Thr
. It is the only site that
was previously identified during the in vitro autophosphorylation studies (Flint, A. J., Paladini, R. D., and
Koshland, D. E., Jr.(1990) Science 249, 408-411). The
fourth peptide Asn
-Lys
is phosphorylated at
Thr
. A discussion of the potential implication of these
results follows.
Phosphorylation is a rapid and reversible means of regulating protein activity. Its efficiency is evident in the many signal transduction pathways that use cascades of phosphorylation to effect cellular responses(1, 2, 3) . Protein kinase C plays a major role in many of these pathways(4, 5, 6) . It is a serine/threonine kinase dependent on calcium and phospholipids and activated by diacylglycerols, fatty acids, or phorbol esters at physiological calcium concentrations(7) . 12 members of the mammalian protein kinase C family have been identified so far(8) . Regions of conservation as well as proteolysis studies indicate that protein kinase C is comprised of two domains, an N-terminal regulatory domain and a C-terminal catalytic domain(9, 10) .
Protein kinase C autophosphorylates itself in vitro on both its regulatory and catalytic domains(11) . Autophosphorylation is particularly intriguing in that it has been shown to be an intramolecular reaction(12) , in which regions very distinct in the primary sequence have access to the active site(13) . When separated from the regulatory domain by proteolysis, the catalytic domain is no longer able to autophosphorylate, even though it is still fully active against substrates(12) .
Six in vitro autophosphorylation sites have been identified in the II
isozyme(13) . Ser
and Thr
are located
close to the autoinhibitory sequence in the primary structure.
Thr
and Thr
are located in the hinge region
between the catalytic and regulatory domains. Thr
and
Thr
are in the C terminus and are the only sites
conserved in all the conventional protein kinase C isozymes. These
residues are outside the region conserved in most other
serine/threonine kinases.
Recent studies in vitro and in vivo have elucidated a definite role for phosphorylation of
protein kinase C. Phosphorylation by a second kinase is thought to be
necessary in the activation of the kinase in
vivo(14, 15) . Mutagenesis studies of Thr and Thr
in protein kinase C isozyme
and
, respectively, have proposed phosphorylation of those residues as
critical for activity in vivo and/or in vitro(15, 16, 17) . In addition, mutations of
the in vitro autophosphorylation sites in protein kinase C
I suggest a role for the C-terminal sites, Thr
and
Thr
in protein kinase C localization, activation, and
down-regulation(18) .
Since previous results indicated that
protein kinase C is phosphorylated in vivo and that
phosphorylation is essential for activation, it is important to
determine whether these phosphorylation sites are identical to in
vitro autophosphorylation sites. The baculovirus expression system
was chosen for protein kinase C expression because of the ease of
purifying a single isozyme. Previous work has shown that the gel
mobility and in vitro autophosphorylation pattern were
identical between protein kinase C II overexpressed in insect
cells and purified from rat brain(13) . In addition,
phosphatase-treated protein kinase C from both sources exhibit similar
gel shifts, suggesting identical phosphorylation patterns(19) .
Mass spectrometry has been successfully used for the determination
of phosphorylation sites in various proteins, such as the chemotaxis
response regulator protein from Escherichia coli(20) ,
bovine myelin basic protein(21) , bovine mitogen-activated
protein kinase(22) , and bleached bovine
rhodopsin(23) . HPLC ()directly coupled with
electrospray ionization mass spectrometry (LC/ESIMS) provides means for
quick and efficient screening of entire protein digests for covalent
modifications(24) . LC/ESIMS analysis yields singly or multiply
protonated peptide ions, and from the m/z value of
these ions, the molecular mass of the corresponding peptide can be
determined. Similarly, liquid secondary ionization mass spectrometry
(LSIMS) analysis usually yields only molecular weight data in the form
of protonated peptide ions. These ions can be activated by collision
with inert gas atoms, such as helium. The dissociation induced this way
usually reveals the amino acid sequence of the peptide analyzed.
Collision-induced dissociation (CID) analysis offers a tool to
determine the amino acid sequence of the peptide and the exact site(s)
of phosphorylation(25) ; while under reaction conditions
required for Edman degradation, the phosphate group is hydrolyzed or
eliminated from phosphorylated serines or threonines. Tandem mass
spectrometry permits CID analysis in mixtures by allowing the precursor
ion selection for collisional activation. Therefore, we used LC/ESIMS
and high energy CID analysis to identify the in vivo phosphorylation sites in protein kinase C
II.
To study the post-translational modifications of protein
kinase C, we expressed protein kinase C II in insect cells. The
tryptic digest of the carboxymethylated protein was analyzed both by
reversed-phase HPLC, followed by LSIMS, and by on-line LC/ESIMS.
Tryptic peptides were identified by comparison of the molecular masses
observed with those predicted from the published sequence(26) ;
more than 90% of the sequence was identified (Fig. 1). The
missing components are small hydrophilic peptides. Post-translational
or other covalent modifications can be indicated by discrepancies
between the predicted and observed molecular masses. For example, the
expected N-terminal tryptic peptide at mass to charge ratio (m/z) 1897.9 was not detected; however, a molecular
mass (1809.0 Da) observed in the LC/ESIMS experiment and later measured
also by LSIMS (MH
at m/z 1808.8)
suggested that the N-terminal methionine had been replaced by an N-acetyl group. This hypothesis has been confirmed by high
energy CID analysis (data not shown). Phosphopeptides were identified
based on the 80-Da mass difference between the predicted and observed
molecular masses. Addition of a phosphate group increases the molecular
mass of a peptide by 80 Da. Four phosphopeptides were identified in the
digest with molecular masses of 1677.4 Da (rt
36 min), 2484.6 Da
(rt
51 min), 3630.0 Da (rt
56.5 min), and 2771.5 Da (rt
57 min), corresponding to phosphorylated sequences
His
-Arg
,
Thr
-Lys
,
Glu
-Lys
, and
Asn
-Lys
, respectively. Fig. 2shows
the HPLC chromatogram of the tryptic digest. No evidence for
phosphorylation on multiple sites on a single tryptic peptide was
detected.
Figure 1:
Molecular weight mapping of protein
kinase C II enzyme. Sequences underlined were detected in
the tryptic digest of the protein either by LSIMS or by LC/ESIMS. Most
of the components were identified by mass
only.
Figure 2:
HPLC
chromatogram of carboxymethylated protein kinase C tryptic digest. The
tryptic peptides (70 pmol) were separated by reversed-phase HPLC
on a Vydac C
, 1.0 mm, inner diameter
250-mm
column. Solvent A was 0.1% trifluoroacetic acid in water, and solvent B
was 0.08% trifluoroacetic acid in acetonitrile. The eluant was
monitored at 215 nm. Phosphopeptide His
-Arg
started to elute in peak 1. Peak 2 contained
both the non-modified and phosphorylated peptides for this sequence.
Peptides corresponding to non-modified and phosphorylated
Thr
-Lys
eluted in peak 3 (See Fig. 5). Phosphopeptide Glu
-Lys
eluted in peak 4. Phosphopeptide
Asn
-Lys
eluted in peak 5. These
species were not fully separated and coeluted with other tryptic
peptides. Peak 6 contained peptide
Asn
-Lys
without any covalent modification. AUFS (absorbance units full scale) gives the relative peak
absorbance.
Figure 5:
Electrospray mass spectrum of protein
kinase C tryptic peptide Thr-Lys
non-modified and phosphorylated. This spectrum was recorded in an
LC/ESIMS experiment from approximately 70 pmol of the tryptic digest.
The average molecular masses of the peptides observed are shown. The
calculated average molecular masses are 2405.7 and 2485.7 Da for the
non-modified and for the phosphopeptide,
respectively.
High energy CID analysis was used to confirm the peptide
sequence and identify the exact site of phosphorylation. High energy
CID processes lead to bond cleavages all along the peptide
backbone(30) . The proton can be retained on either of the
newly formed species, yielding an ionic and a neutral fragment; the
mass spectrometer only detects ionized molecules. Ions with charge
retention at the N terminus are designated as a, b, and c ions, while those at the C terminus are designated as x, y, and z ions, respectively. Fragment ions a and x are the products of a bond cleavage between the
-carbon and the carbonyl group. Ions b and y are
formed from cleavage of the peptide bond itself. Fragments c and z are generated when the cleavage occurs between the amino group
and the
-carbon. Fragment ions v, w, and d are formed by a backbone and a side chain cleavage, with charge
retention on the C or the N terminus,
respectively(30, 31) . The expected mass values of the
fragments can be calculated for peptides with known amino acid
sequence.
The peptide of molecular mass of 1677.6 Da was subjected
to high energy CID analysis, which confirmed the amino acid sequence as
His-Arg
and the presence of a phosphate
group at Thr
(Fig. 3). This peptide was observed
without modification as well (rt
37 min). Based on the relative
ion abundances of the phosphorylated and non-modified peptides from
LC/ESIMS analysis and in vitro autophosphorylation
studies(13) , it is estimated that this site is phosphorylated
at least 75%.
Figure 3:
High energy CID spectrum of phosphorylated
peptide His-Arg
. MH
= 1677.8. Fragment ions are labeled according to the
accepted nomenclature(40) .
Since both peptides Glu-Lys
and Thr
-Lys
were observed with a
molecular mass increase of 80 Da, and peptide
Glu
-Lys
was observed only without the
phosphate group, it can be deduced that the modification occurs either
on Thr
or Thr
. Phosphopeptide
Glu
-Lys
was subjected to digestion with
various enzymes to produce smaller peptides more suitable for high
energy CID experiments. The peptide proved to be resistant to
chymotrypsin, and endoproteinase Glu-C removed only the C-terminal nine
amino acids. Digestion with endoproteinase Asp-N eventually yielded a
phosphopeptide in the desired molecular weight range,
D
GVTTKTFC*GTP
with MH
at m/z 1364.6, which was then subjected to high energy
CID analysis (Fig. 4). Fragment ions with charge retention at
the N terminus for the first six residues do not indicate the presence
of any covalent modification. However, N-terminal fragment ion a
(at m/z 755) that results from a
cleavage between the
carbon of Thr
and its carbonyl
group exhibits an 80-Da mass shift, corresponding to a phosphate group.
Similarly all the other N-terminal ions containing Thr
display this 80-Da mass shift. C-terminal ion y
, which is formed via peptide bond cleavage between
Gly
and Thr
with charge retention at the C
terminus, was detected at m/z 217, thus indicating no
covalent modification at Thr
. Thus, the modification
occurred at Thr
. A peptide for
Thr
-Lys
with no modification was detected
as a minor component in the LC/ESIMS experiment (rt
51 min, Fig. 5). Peptide Glu
-Lys
was only
detected with the modification. The site occupancy for Thr
is estimated to be higher than 90%.
Figure 4:
High energy CID spectrum of phosphorylated
peptide Asp-Pro
. MH
= 1364.6. N-terminal sequence ions starting from the amino
acid at position 7 (Thr
) show the 80-Da mass shift. The
cysteine marked with an asterisk is carboxymethylated. Ions
labeled with asterisks are matrix-related background
ions(41) .
The identity of
phosphopeptide Asn-Lys
was confirmed by
Edman degradation (see Table 1). The mass is increased by a
single 80-Da increment, indicating one phosphate group per peptide.
Since the peptide contains three possible phosphorylation sites,
Ser
, Ser
, and Ser
, attempts
were made to produce peptides containing individual phosphorylation
sites. The peptide was resistant to endoproteinases Glu-C and Asp-N;
endoproteinase Asp-N was tried since it was reported to cleave at the N
terminus of not only aspartic acids but also at the N terminus of other
negatively charged residues such as cysteic acids (32) and
glutamic acids(24) . Chymotryptic digestion yielded a
phosphopeptide, Asn
-Phe
, still containing
all three serine residues. Based on the UV and LC/ESIMS data, the
occupancy of on peptide Asn
-Lys
is
estimated to be greater than 80%. Keranen, Dutil, and Newton have
informed us (
)that the phosphorylation occurs at
Ser
. This correlates well with the high degree of
conservation of Ser
in comparison with Ser
and Ser
(Fig. 6).
Figure 6:
Sequence alignment of phosphopeptide,
Asn-Lys
, of protein kinase C
II with
seven other members of the protein kinase C family. Potential
phosphorylation sites Ser
, Ser
, and
Ser
and residues aligned with them are shaded.
Notably, this phosphopeptide falls within a defined variable
region.
Three distinct sites of phosphorylation, Thr,
Thr
, and one of the serines on phosphopeptide,
Asn
-Lys
, were determined by mass
spectrometry (Fig. 7). Each site was phosphorylated greater than
75%. Thr
lies in the conserved serine/threonine kinase
catalytic region. Thr
lies outside that conserved region,
but the residue itself is conserved in the protein kinase C family.
Phosphopeptide Asn
-Lys
is at the C terminus
and lies within a region defined as variable among the major members of
the protein kinase C family.
Figure 7:
Linear representation of the primary
sequence of protein kinase C II, indicating the location of
phosphorylation sites. In vivo sites or phosphopeptides,
identified by mass spectrometry, are marked by arrows. In
vitro autophosphorylation sites are shown in the filled
circles. Hatched regions indicate the areas conserved in
the protein kinase C family. The region conserved with other
serine/threonine kinases is marked by the bracket.
Of the autophosphorylation sites
previously identified in vitro(13) , only Thr is detected in this analysis of unstimulated sample. The fact
that 75% of the sample is already phosphorylated at this residue
explains the apparent low labeling level detected in the in vitro autophosphorylation studies. A single mutation to alanine at the
corresponding residue in the
I isozyme decreases activity in
vivo, and the mutant is no longer able to
autophosphorylate(33) . Recent work using phosphatase treatment
and subsequent autophosphorylation of protein kinase C
II has
suggested that protein kinase C is solely responsible for
phosphorylation of this residue(19) .
It is notable that
only one of six in vitro autophosphorylation sites was found
to be phosphorylated in this study. The difference between the level of
protein kinase C stimulation in vitro (activation by
diacylglycerol, Ca, and phosphatidylserine) (13) and in vivo (no artificial stimulation) could
explain a lack of autophosphorylation. However, one of the sites,
Thr
, is phosphorylated, which suggests that protein
kinase C was activated in vivo. If protein kinase C
autophosphorylates itself at Thr
, why are the other
autophosphorylation sites also not phosphorylated? Possible
explanations are (a) degree of accessibility of Thr
relative to the other sites, (b) sensitivity or
resistance to phosphatases, (c) specific post-translational
processing of protein kinase C(19) , (d) a second
kinase phosphorylating only Thr
(33) , or (e) discrepancies between the plasma membrane in vivo and detergent micelles in vitro. Further work will be
needed to clarify this issue.
The observed phosphorylation of
protein kinase C at Thr is interesting in view of known
serine/threonine kinase structures. In protein kinase A(34) , a
phosphorylated residue at this position is necessary for the integrity
of the active site structure. In the modeled protein kinase C
structure, a phosphorylated threonine would be able to interact with
surrounding residues in a manner very reminiscent to protein kinase A (17) . These residues are conserved in many other
serine/threonine kinases(35) . Complete in vivo phosphorylation at Thr
supports the conclusion that
it is the activating phosphorylation site conserved in many
kinases(36) .
Phosphorylation at Thr agrees
with biochemical evidence that this residue is critical for activity.
Mutagenesis studies demonstrated that Thr
and Thr
in the
and
II isozymes, respectively, were essential
for activity(16, 17) . The authors suggested that a
second kinase must be phosphorylating and thus activating protein
kinase C. In fact, replacement of Thr
in the
II
isozyme with glutamate restored complete activity and suggests that it
is the phosphorylation of the residue that is critical(17) .
Thorsness and Koshland (37) have shown that an aspartate can
mimic the presence and an alanine the absence of an inhibitory
phosphorylation in isocitrate dehydrogenase. In protein kinase C, it
appears that the larger glutamate is better able to maintain the
integrity of the active site. These studies are in agreement with our
identification of greater than 90% in vivo phosphorylation at
Thr
.
Deletion studies of protein kinase C (38) suggest that phosphorylation near the C terminus is
critical for protein kinase C activity. Truncation of 23 amino acids
from the C terminus fully inactivates the kinase(38) ;
Ser
corresponds to the 16th residue from the C terminus
in the
isozyme (Fig. 6). In addition, the high degree of
conservation of Ser
suggests a potential family-wide
regulation (Fig. 6).
We have determined in vivo phosphorylation sites of unstimulated protein kinase C II.
All three of these regions appear to play a strong role in protein
kinase C function. Phosphorylation at a particular residue such as
Thr
may be of structural importance. The other
phosphorylated sites may be involved in substrate recognition or
activator affinity. The fact that activators of protein kinase C
increase the phosphorylation state while epidermal growth factor
decreases the phosphorylation state (39) suggests that
phosphorylation is an important means of regulating protein kinase C
activity.