(Received for publication, September 22, 1994; and in revised form, December 7, 1994)
From the
Two cytosolic proteins of murine epidermis or porcine spleen
with molecular masses of 37 kDa (p37) and 50 kDa (p50) are
differentially phosphorylated in vitro by the purified protein
kinase C (PKC) isoenzymes ,
,
(cPKC) and PKC
. p37,
identified as annexin I, is preferentially phosphorylated by cPKC,
whereas p50, identified as elongation factor eEF-1
, is
phosphorylated with much greater efficacy by PKC
than by cPKC.
Using the recombinant PKC isoenzymes
,
,
,
,
,
, and
, we could show that purified eEF-1
is indeed a
specific substrate of PKC
. It is not significantly phosphorylated
by PKC
, -
, and -
and only slightly by PKC
, -
,
and -
. PKC
phosphorylates eEF-1
at Thr-431 (based on the
murine amino acid sequence). The peptide RFAVRDMRQTVAVGVIKAVDKK with a
sequence corresponding to that of 422-443 from murine eEF-1
and containing Thr-431 is an absolutely specific substrate for the
-type of PKC. The single basic amino acid close to Thr-431
(Arg-429) is essential for recognition of the peptide as a substrate by
PKC
and for the selectivity of this recognition. Substitution of
Arg-429 by alanine abolishes the ability of PKC
to phosphorylate
the peptide, and insertion of additional basic amino acids in the
vicinity of Thr-431 causes a complete loss of selectivity.
The PKC ()family, a group of 11 isoenzymes known so
far possessing phospholipid-dependent serine/threonine kinase activity,
plays a key role in cellular signal transduction and is involved in the
regulation of numerous cellular processes and probably in tumor
promotion (for reviews, see (1, 2, 3, 4) ). The PKC members can
be subdivided into three groups. The conventional PKCs (cPKCs;
,
,
, and
) are
Ca
-responsive and activated by diacylglycerol or
tumor-promoting phorbol esters such as
TPA(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) ;
the novel PKCs (nPKCs;
,
,
, and
) are
also activated by diacylglycerol or TPA but are
Ca
-unresponsive(1, 2, 3, 4, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) ;
the atypical PKCs (aPKCs;
,
, and
) are
Ca
and diacylglycerol- and
TPA-unresponsive(1, 2, 3, 4, 12, 26, 27, 28, 29, 30, 31, 32, 33) .
PKC isoenzymes are differentially expressed and respond differently to physiological stimuli in various tissues and cell types (for review, see (34) ). Therefore, it is likely that they serve different physiological purposes. Indeed, overexpression of various PKCs in different cell lines indicated that certain isoenzymes might induce a specific effect such as increased or reduced cell proliferation(35, 36, 37, 38, 39, 40) . Other experimental devices, such as overexpression of kinase-negative mutants, gene knock-out, or a permeabilization system to reconstitute PKC responses also provided some insight into cellular effects specifically induced by PKC isoenzymes(34) . However, the distinct functions of different isoenzymes in signaling processes, especially with respect to selective phosphorylation of substrates, are unknown to date. Some differences in the phosphorylation of a few commonly used PKC substrates by purified or partially purified PKC isoenzymes in vitro were observed previously (16, 17, 19, 20, 24, 26, 31, 32, 41). Furthermore, a few physiological substrates (42, 43, 44, 45, 46) were shown to be phosphorylated differentially by various PKC isoenzymes. Selective phosphorylation of a substrate by a single PKC isoenzyme has not been reported as yet.
Here we show that the elongation factor
eEF-1 as well as an eEF-1
peptide are phosphorylated
selectively by the
-type of PKC isoenzymes and that the amino acid
environment of the phosphorylation site, i.e. Thr-431,
determines this selectivity.
Purification of
PKC and cPKC was performed as described elsewhere (15, 47) .
The amino acid sequence of phosphopeptides was determined by automated Edman degradation in an Applied Biosystems, Inc. (Foster City, CA) model 473A protein sequencer. Phosphothreonine was identified as described by Meyer et al.(49) .
SDS-polyacrylamide gel electrophoresis was performed on 9% gels, and protein content was determined as described previously(50) .
Figure 1:
Chromatography of cytosol from porcine
spleen on Q-Sepharose and differential phosphorylation of p37 and p50
in the void volume by cPKC and PKC. Chromatography and
phosphorylation of proteins (40 µg) were performed as described
under ``Methods.'' Phosphorylated proteins were separated by
SDS-polyacrylamide gel electrophoresis and visualized by
autoradiography.
Figure 2:
Purification of p50 from the Q-Sepharose
void volume by chromatography on S-Sepharose (see
``Methods''). Purified p50 in fractions 11, 12, and 13 of the
S-Sepharose eluate (NaCl gradient; 30-µl aliquots) was
phosphorylated with PKC run on an SDS-polyacrylamide gel and
visualized by autoradiography and Coomassie Blue
staining.
Figure 3:
Identification of p50 as eEF-1.
Comparison of purified p50 with authentic eEF-1
by immunoblotting
with an anti-eEF-1
antiserum and by autoradiography of the
PKC
-phosphorylated proteins. Alkaline phosphatase-conjugated goat
anti-rabbit IgG was used for immunostaining. Immunoblot: 10
µg of p50, 0.4 µg of eEF-1
; Autoradiogram: 10
µg of p50, 0.2 µg of eEF-1
. A limited amount only of
authentic eEF-1
was available. The intensity of eEF-1
in the
immunoblot was found to be around 30% and in the autoradiogram around
8% of that of p50 and thus more than expected. This was probably due to
a nonlinear relationship between the amount of protein and the
intensity of either immune reaction or
autoradiography.
The differential
phosphorylation of eEF-1 by cPKC and PKC
was studied in more
detail. Fig. 4shows the time dependence of the eEF-1
phosphorylation by cPKC and PKC
. After 60 min, when saturation was
reached, eEF-1
had incorporated around 7 times more phosphate in
the presence of PKC
(0.4 mol of phosphate/mol of eEF-1
) than
in the presence of cPKC. The K
and V
values of the phosphorylation by PKC
were
0.21 µM and 4.21 nmol of phosphate/min/mg and of the
phosphorylation by cPKC 0.27 µM and 0.37 nmol of
phosphate/min/mg, respectively (Table 1). Thus, the concentration
of eEF-1
in a tissue (about 20 µM) is 100-times greater than
the K
. The V
/K
value of the
phosphorylation by PKC
was about 15 times higher than that by
cPKC. The finding that eEF-1
was selectively phosphorylated by
PKC
became even more evident using various recombinant PKC
isoenzymes (
,
,
,
,
,
, and
) from
baculovirus-infected insect cells. The isoenzymes were used at equal
activities based on phosphorylation of either protamine sulfate or
myelin basic protein. In both cases, PKC
was by far the most
effective isoenzyme (Fig. 5). Other
Ca
-unresponsive isoenzymes (
,
, and
)
did not phosphorylate eEF-1
, whereas the
Ca
-responsive isoenzymes (
,
, and
),
in accordance with the results obtained with cPKC, phosphorylated
eEF-1
slightly. The extremely intense autophosphorylation of
PKC
(see Fig. 5) has previously been observed with the
purified enzyme from porcine spleen, which incorporated around 4 times
as much phosphate as cPKC (15) . (
)
Figure 4:
Time dependence of eEF-1
phosphorylation by cPKC and PKC
. 2.5 µg of eEF-1
were
phosphorylated with 8 milliunits of each of cPKC (
) or PKC
(
) for the times indicated as described under
``Methods.''
Figure 5:
Phosphorylation of eEF-1 by various
recombinant PKC isoenzymes. 2.5 µg of eEF-1
were
phosphorylated with extracts of baculovirus-infected insect cells
expressing PCK
,
,
,
,
, or
, as
described under ``Methods.'' Equal activities of the
isoenzymes were used as determined by phosphorylation of myelin basic
protein or protamine sulfate. Depending on the isoenzyme's
activity, the extracts added to the assay contained 5-25 µg
of protein. PKC
was used only in one of the two experiments (upperpart of the
figure).
Figure 6:
Separation of tryptic eEF-1 peptides
by HPLC and determination of amino acid sequences of phosphorylated
peptides. PKC
-phosphorylated eEF-1
was digested with trypsin;
tryptic peptides were separated by two HPLC runs (first run, A; second run, B); and the amino acid sequence of the
phosphorylated peptides A and B were analyzed as described under
``Methods.'' OD215 (-), acetonitrile
(
), cpm
(-
-
-
).
Figure 7:
Phosphorylation of the eEF-1 petide
(petide 1) by 8 milliunits each of cPKC (
) or PKC
(
).
For details of the procedure, see
``Methods.''
Figure 8:
Phosphorylation of the eEF-1 peptide
1 and the mutated petides 3, 4, and 5 (5 nmol each) by recombinant PKC
isoenzymes (see Fig. 5). Equal activities of the isoenzymes
based on the phosphorylation of myelin basic protein were used. Peptide 1: RFAVRDMRQTVAVGVIKAVDKK; peptide 3:
RFAVRDRMQTVAVGVIKAVDKK; peptide 4: MFAVRDRRQTVAVGVIKAVDKK; peptide 5: MFAVRDRRQTVKKGVIKAVDAV.
In the cell, the different PKC isoenzymes are thought to have
a distinct function regarding phosphorylation of specific substrate
proteins. Selective substrate phosphorylation could be due to an
intrinsic specificity of an isoenzyme and/or to a specificity acquired
by modification of an isoenzyme (e.g. by phosphorylation)
and/or to a specific localization of an isoenzyme. Very recently, we
have shown that PKC is phosphorylated in vitro by Src and that this tyrosine phosphorylation induces a stimulation of
PKC
activity apparently exhibiting some substrate
selectivity(51) . Specific intracellular localizations of
different PKC isoenzymes were reported, but a correlation with
selective substrate phosphorylation has not been demonstrated as yet.
Various purified or partially purified PKC isoenzymes were tested in vitro for substrate selectivity, and some differences in
the phosphorylation of substrates were observed. Most frequently,
however, only histone, myelin basic protein, protamine sulfate, and
pseudosubstrate-related peptides were used as substrates (16, 17, 19,
20, 24, 26, 31, 32, 41). There are a few reports on other substrates,
such as glycogen synthase kinase-3
(42) , epidermal growth
factor receptor(43) , vitamin D receptor(44) ,
neuromodulin(45) , and Raf(46) , which appear
to be preferentially phosphorylated in vitro by one or the
other PKC isoenzyme. However, selective phosphorylation of a substrate
by a PKC isoenzyme has not been reported as yet.
We tried to find
isoenzyme-specific substrate proteins in tissue extracts and were able
to show that a 50-kDa protein in the cytosol of murine epidermis or
porcine spleen is phosphorylated to a much greater extent by PKC
than by cPKC (PKC
, -
, and -
). The significance of this
observation is supported by the concomitant finding that another
protein (p37, identified as annexin I) is phosphorylated much more
efficiently by cPKC than by PKC
. This indicates the existence of a
differential phosphorylation of some proteins by different PKC
isoenzymes. p50 could be identified as the elongation factor eEF-1
by comparison with authentic eEF-1
and by immunoblotting with an
anti-eEF-1
antibody. Moreover, the sequence of a tryptic peptide
of p50 is identical with the sequence 428-438 of eEF-1
(see
below). According to our results obtained with various recombinant PKC
isoenzymes from baculovirus-infected insect cells, purified eEF-1
is most effectively phosphorylated by PKC
, only slightly by
PKC
, -
, and -
, and not at all by PKC
, -
, and
-
. The amount of phosphate incorporated into eEF-1
by
PKC
indicates the existence of only one phosphorylation site, and,
according to the phosphoamino acid analysis, this single site is a
threonine. In accordance with these results, sequence analysis of
purified tryptic peptides of phosphorylated eEF-1
revealed that
Thr-431 is the sole amino acid in eEF-1
, which is phosphorylated
by PKC
. The physiological significance of this phosphorylation is
not known as yet. Phosphorylation of eEF-1
by a partially purified
PKC preparation (probably a mixture of PKC
, -
, and -
) in vitro and in intact cells upon treatment with TPA was
reported previously(52, 53) . Phosphorylation in
vitro of the subunit eEF-1
alone, i.e. not complexed
with the other subunits of eEF-1
(
and
) did not affect
its translation activity. However, besides its role as elongation
factor in protein biosynthesis, eEF-1
appears to exhibit still
other functions (for review, see (54) ). It was shown to play a
role in cell cycle-associated processes(55, 56) , in
ubiquitin-regulated degradation of proteins(57) , in
aging(58, 59) , and as a regulator of cytoskeletal
rearrangements(56, 60, 61) . It is
conceivable that some of these activities of eEF-1
are modified by
its phosphorylation.
Knowing the phosphorylation site, we became
interested in the question as to whether the amino acid environment of
Thr-431 was responsible for the PKC-specific phosphorylation of
Thr-431 and whether this isoenzyme specificity could be demonstrated in
an appropriate synthetic peptide. Most intriguingly, a peptide
corresponding to sequence 422-443 of murine eEF-1
and
containing Thr-431 (peptide 1) is phosphorylated with high specificity
by PKC
, i.e. other isoenzymes do not accept this peptide
as a substrate. It is conceivable that this peptide may be used to
selectively determine PKC
activity in crude tissue or cell
extracts. This possibility is presently being investigated. It is well
known that phosphorylation sites of PKC substrates require basic amino
acids in the vicinity of serine or threonine that become
phosphorylated. Consensus phosphorylation site motifs for PKC were
defined, such as (K/R)XX(S/T), (K/R)X(S/T),
(K/R)XX(S/T)X(K/R), etc.(62) . Turner et
al.(63) reported on the differential phosphorylation of
synthetic peptides derived from bovine myelin basic protein and some
mutated forms of these peptides by a PKC preparation from pig brain
(probably a mixture of PKC
, -
, and -
). Marais et al.(64) performed a systematic investigation of sequence
requirements for synthetic peptides functioning as substrates for
PKC
, -
, and -
. However, respective studies
with isoenzymes of the nPKC group have not been reported, and a peptide
with an amino acid sequence recognized selectively by a PKC isoenzyme
has not been described as yet. The eEF-1
peptide with the
PKC
-specific phosphorylation site contains only 1 basic amino acid
(Arg-429) in close vicinity to Thr-431. This is in accord with
(K/R)X(S/T) being one of the consensus phosphorylation site
motifs for PKC(62) . Upon removal of this basic amino acid, the
peptide (peptide 2) is no longer able to serve as a substrate for
PKC
. Even a slight shift of this basic amino acid just one
position away from the phosphorylation site (peptide 3) causes a
significant decrease of the phosphorylation by PKC
. This clearly
demonstrates that the minimal requirement of PKC
for the
phosphorylation site in the eEF-1
peptide is the presence of a
single basic amino acid close to the phosphate-accepting amino acid.
Obviously, the minimal requirement of the other PKC isoenzymes for this
phosphorylation site is different since they do not recognize the
eEF-1
peptide as a substrate. We assume that phosphorylation sites
of the other isoenzymes might require more than 1 basic amino acid.
Indeed, a mutated eEF-1
peptide with 2 basic amino acids on either
side of Thr-431 (428-429 and 433-434; peptide 5) is
phosphorylated by all PKC isoenzymes. Moreover, the mutated peptide is
phosphorylated by PKC
much more efficiently than the original
peptide 1. Surprisingly, a mutated eEF-1
peptide with 2 basic
amino acids only on one side of Thr-431 (428-429; peptide 4) does
not show the drastic increase in phosphorylation by all PKC isoenzymes
that is observed with peptide 5. This indicates that PKC
, like the
other PKC isoenzymes, prefers phosphorylation sites with a strong basic
environment but, at least with some substrates, tolerates weak basic
sites better than the other isoforms. Probably due to this property,
PKC
is able to phosphorylate eEF-1
and possibly also other
substrates selectively.