(Received for publication, November 6, 1995; and in revised form, December 27, 1995)
From the
The respiratory burst oxidase is responsible for superoxide
(O) production by phagocytes and B
lymphocytes. This multicomponent enzyme is dormant in resting cells but
is activated on exposure of the cells to an appropriate stimulus. Upon
activation, several serine residues on the cytosolic oxidase subunit
p47
become phosphorylated. Using
two-dimensional tryptic phosphopeptide mapping, we studied the
phosphorylation of p47
in
P
-loaded Epstein-Barr virus-transformed B
lymphoblasts expressing wild type p47
or any of
several p47
Ser
Ala mutants. We were
able to identify the labeled peptides from wild type p47
as those containing Ser
,
Ser
, Ser
, Ser
and/or
Ser
, and Ser
; no
P-labeled Ser
-containing peptide was found.
When purified p47
was phosphorylated in
vitro by various protein kinases, varying phosphopeptide patterns
were observed. Protein kinase C phosphorylated all the peptides except
the one containing Ser
; protein kinase A
phosphorylated the peptide containing Ser
and one or both
of the peptides containing Ser
and Ser
;
while mitogen-activated protein kinase phophorylated only the peptide
containing Ser
. These findings suggest that these
three kinases play distinct roles in the activation of the respiratory
burst oxidase, each of them catalyzing the phosphorylation of a
different group of serines in p47
.
The respiratory burst oxidase of phagocytes and B lymphocytes
catalyzes the reduction of oxygen to superoxide
(O) at the expense of
NADPH(1, 2, 3, 4, 5, 6) .
In resting cells the enzyme is inactive, and its components are
distributed between the cytosol and the membranes of secretory
vesicles. When the cells are activated, the cytosolic components
migrate to the membranes, where they associate with the membrane-bound
components to assemble the catalytically active oxidase (1, 5, 7) .
When the oxidase is activated,
p47, one of the cytosolic subunits, becomes
phosphorylated on several serines(8, 9) . We recently
found that in human neutrophils serines Ser
,
Ser
, Ser
, Ser
,
Ser
, Ser
, and Ser
and/or
Ser
are phosphorylated and that other serines lying
between Ser
and Ser
could be
phosphorylated(10) . We further showed that at least one of
these serines is absolutely required for oxidase activation in whole
cells stimulated with PMA(11) . In this study, we report the
use of site-directed mutagenesis combined with two-dimensional
phosphopeptide mapping to further characterize the phosphorylation of
p47
in B lymphocytes and to compare the in
vitro phosphorylation of purified p47
by
various serine/threonine-specific protein kinases.
Our
approach was to express p47 Ser
Ala mutants in
EBV-transformed p47
-deficient B lymphocytes; to load
these lymphocytes with
P
and then activate
them with PMA to label their p47
; and finally to purify
the labeled p47
mutants, map them, and look for
differences between those maps and the map of
P-labeled WT
p47
. In a tryptic digest of p47
, the
phosphorylation targets are distributed among several peptides (Table 1; trypsin is unable to split Lys-Pro and
Arg-Pro bonds)(17) . Of these, the peptide containing
Ser
and Ser
would probably be difficult to
see because it would contain very little
P relative to the
other peptides(10, 11) . The results (Fig. 1, left) showed that the map of WT p47
contained
six major phosphopeptides (arrows), all of which were seen in
each of 15 separate experiments, together with several minor
phosphopeptides whose presence in the maps was inconstant. Taking into
consideration the serines known to be phosphorylated during oxidase
activation (10) and the peptides generated by tryptic digestion
of p47
(Table 1), we made a number of mutants in
which two or more serines had been converted to alanines, and used
these together with mutants containing a single Ser
Ala change
to identify the labeled peptides on the two-dimensional map.
Figure 1:
Phosphopeptide maps of
p47isolated from PMA-activated
p47
-deficient B lymphocytes expressing WT and
mutant forms of p47
. Labeling and activation of
EBV-transformed p47
-deficient B lymphoblasts
expressing WT and mutant p47
, isolation and
purification of the labeled p47
, and
phosphopeptide mapping were carried out as described under
``Experimental Procedures.'' The mutations are indicated in a
corner of each panel; 47S = WT. The point of
application of the sample is indicated by the dot in the lower left corner of each panel. Missing peptides are outlined
with dotted lines. Left, mutations that produce a change in
the phosphopeptide map. Arrows show the major phosphorylated
peptides. Right, mutations that have no effect on the
phosphopeptide map.
Restricting our analysis to the six constant phosphopeptides (Fig. 1), we found that maps of p47 mutants
containing single Ser
Ala mutations were the same as WT maps (Fig. 1, left) except for the map of S320A, which
lacked a single spot, and the map of S315A, which lacked two spots (Fig. 1, left). The latter result suggests that at
least two peptides were produced, probably because of partial cleavage
at the sequence RKR (residues 316-318). Sequences containing
basic residues in tandem are known to be susceptible to partial
cleavage(17) .
Certain of the peptides of interest contain
two serines, and it may be that the elimination of both serines is
necessary to eliminate a spot corresponding to such a peptide. In
accord with this idea, a single spot was eliminated from the
phosphopeptide maps of S303A,S304A and S345A,S348A (Fig. 1, left). The map of the sextuple mutant S328A-S379A lacked
two spots: one known to correspond to the peptide
Ser, and another that by elimination has to represent
the two peptides Ser
and Ser
, because
the spots corresponding to the remaining phosphopeptides (i.e. Ser
, Ser
, and Ser
)
were present on the map. (
)It appears that the
phosphorylated peptides Ser
and Ser
coincide on the tryptic peptide map. Finally, these results
suggest that Ser
is not phosphorylated during oxidase
activation.
A diagram of the tryptic peptide map of WT p47 giving the identities of the major peptides is shown in Fig. 2. This diagram also shows the location of
P
, which proved to be a useful marker for
identifying peptides on maps with missing spots.
P
lies at a level between peptides Ser
and
Ser
, and migrates toward the anode, while the
phosphopeptides all migrate toward the cathode. The location of the
P
spot relative to the point of application of
the sample (marked by a plus sign (+) in the lower left corner of the figure) provides a mobility standard that allows the
identification of individual peptides even in the absence of
information concerning the overall distribution of spots on the
chromatogram, as would occur under conditions in which only one or two
peptides are phosphorylated.
Figure 2:
A diagram of the phosphopeptide map of
p47 showing the locations and identities of the
major peptides. The major phosphopeptides (filled areas) are
identified by the target serines they contain. The location of the
32P
spot is also shown. Open areas represent
inconstant phosphopeptides on the autoradiogram from which this figure
was derived. The plus sign (+) indicates the point of application
of the sample. On the electrophoresis axis, the cathode is on the right. TLC, thin layer
chromatography.
Figure 3:
Phosphopeptide maps of
p47isolated from activated neutrophils and B
cells. Labeling and activation of EBV-transformed
p47
-deficient B lymphoblasts expressing WT
p47
and of human neutrophils, isolation and
purification of the labeled p47
and
phosphopeptide mapping were carried out as described under
``Experimental Procedures.'' The point of application of the
sample is indicated by the dot in the lower left corner of each panel.
Figure 4:
Phosphopeptides produced by CNBr cleavage
of p47 purified by immunoprecipitation from
human neutrophils phosphorylated with protein kinase C, protein kinase
A, or MAP kinase. The experiment was carried out as described under
``Experimental Procedures'' using p47
purified from human neutrophils. The arrow shows
the location of the C-terminal CNBr fragment of
p47
.
Figure 5:
Phosphopeptide maps of
p47phosphorylated with protein kinase C (top), protein kinase A (middle), and MAP kinase (bottom). The experiment was carried out as described under
``Experimental Procedures'' using p47
purified from human neutrophils. The point of application of
the sample is indicated by the dot in the lower left
corner of each panel.
Figure 6:
Phosphorylation of phosphopeptide 359/370
by protein kinase C and protein kinase A. The experiment was carried
out as described under ``Experimental Procedures.'' A labeled
band at 4 kDa is seen in both tracks.
On the
phosphopeptide maps of purified p47 that had been
labeled with a known kinase, major spots were seen that were not
present on the maps of p47
labeled in whole cells. These
spots were disregarded as irrelevant to the physiological labeling
pattern of activated p47
.
Tryptic peptide mapping of p47 labeled with
P either in intact cells or in a cell-free system provides
an efficient way of identifying which of the target peptides are
phosphorylated, and in combination with image analysis of radioactivity
could yield important information on the relative quantities of
phosphate on various of the serines of the protein. The results
obtained by tryptic peptide mapping retain a certain amount of
ambiguity, however, because they provide no information as to which of
the two serines on a two-serine peptide is (are) phosphorylated.
Whether it is important to answer that question will depend on studies
correlating structure and function in Ser
Ala mutants of
p47
, although a partial answer is provided by our recent
report showing that Ser
Ala mutations of individual serines from
position 303 to 370 have little effect on oxidase
activity(11) .
The present results provide some information
as to the order of phosphorylation of the target serines on
p47. Except for the mutant S315A, whose anomalous
properties were discussed above, mutations affecting the serines on a
single tryptic peptide caused the loss of at most one spot on the
phosphopeptide map. This finding suggests that there is no target
serine whose phosphorylation is absolutely dependent on the
phosphorylation of a serine on a different peptide, or the
phosphorylation of a group of such serines. Rather, it appears that
these serines can be phosphorylated in any order.
We previously
showed that when the respiratory burst oxidase is activated, serines
Ser, Ser
, Ser
,
Ser
, Ser
, Ser
, Ser
and/or Ser
, and Ser
of p47
are phosphorylated(10, 11) . The present studies
confirm the earlier results by another method, and in addition have
shown that Ser
is also phosphorylated, bringing the total
number of phosphorylated serines in the C-terminal region of activated
p47
to 9 or 10. Ser
has already been found
to play an important role in oxidase activation and p47
translocation, and it is likely that protein kinase-mediated
phosphorylation of other target serines is equally important. In fact,
several lines of evidence already support a role for protein kinase C
in oxidase activation. For example, PMA, an activator of several forms
of protein kinase C, is a powerful stimulator of
O
production in whole
cells(20) . Purified p47
is a good substrate for
protein kinase C in vitro(21) , while staurosporine, a
potent inhibitor of protein kinase C (and other kinases), blocks
PMA-induced O
generation as well as the
phosphorylation of p47
(22, 23) .
Finally, we show in this study that the phosphopeptide map of
p47
isolated from PMA-activated neutrophils and
EBV-transformed B lymphocytes is identical to the phosphopeptide map of
p47
phosphorylated in vitro by protein kinase
C, except for the absence of the Ser
peptide from the
latter map. These findings suggest that one or more of the
PMA-responsive forms of protein kinase C could be a critical mediator
of oxidase activation. We showed recently that Ser
and
Ser
are not required for oxidase activation(11) ,
since the S345A,S348A mutant of p47
is fully active in
EBV-transformed B cells. This finding suggests that, in contrast to
protein kinase C, phosphorylation of p47
by
proline-directed kinases such as MAP kinase may have little to do with
oxidase activation. The role of target serines in the regulation of
oxidase activity is currently under investigation in our laboratory.
Phosphorylation of p47 was also shown to occur upon
addition of dibutyryl cAMP to neutrophil cytoplasts or cytosol,
suggesting that p47
is also a substrate for protein
kinase A(24) . Dibutyryl cAMP did not induce
O
production, however, indicating that
phosphorylation by protein kinase A alone is not sufficient to activate
the oxidase. It is possible, in fact, that the the phosphorylation of
p47
by protein kinase A prevents the assembly of the
oxidase, since the elevation of neutrophil cAMP inhibits
O
production(25) . Our results
show that protein kinase A phosphorylates fewer target serines than
protein kinase C, phosphorylating only peptides Ser
,
Ser
, and possibly Ser
(a maximum of
four target serines), in contrast to the five peptides (up to seven
target serines) phosphorylated by protein kinase C. The protein kinase
A targets could be responsible for the negative regulation of
p47
phosphorylation and oxidase activation by protein
kinase A.