(Received for publication, June 26, 1995)
From the
As potential targets for polyphosphoinositides, activation of
protein kinase C (PKC) isotypes (,
,
,
) and a member of the PKC-related kinase (PRK) family, PRK1, has
been compared in vitro. PRK1 is shown to be activated by both
phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P
) as
well as phosphatidylinositol 3,4,5-trisphosphate
(PtdIns-3,4,5-P
) either as pure sonicated lipids or in
detergent mixed micelles. When presented as sonicated lipids,
PtdIns-4,5-P
and PtdIns-3,4,5-P
were equipotent
in activating PRK1, and, furthermore, sonicated phosphatidylinositol
(PtdIns) and phosphatidylserine (PtdSer) were equally effective. In
detergent mixed micelles, PtdIns-4,5-P
and
PtdIns-3,4,5-P
also showed a similar potency, but PtdIns
and PtdSer were 10-fold less effective in this assay. Similarly,
PKC-
, -
, and -
were all activated by
PtdIns-4,5-P
and PtdIns-3,4,5-P
in detergent
mixed micelles. The activation constants for PtdIns-4,5-P
and PtdIns-3,4,5-P
were essentially the same for all
the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of
PtdIns-4,5-P
and PtdIns-3,4,5-P
were found to
be inhibited at 10 mM Mg
and mimicked by
high concentrations of inositol hexaphosphate and inositol hexasulfate.
The similar responses of these two classes of lipid-activated protein
kinase to these phosphoinositides are discussed in light of their
potential roles as second messengers.
The phosphatidylinositol 3-kinase family of lipid kinases are
responsible for the phosphorylation of inositol lipids at the 3-OH
position (reviewed in (1) ). In response to various agonists,
phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P) (
)accumulates and labeling studies suggest that this is the
primary product and as such is the most likely second messenger
candidate(2) . While this remains an attractive hypothesis, in
the absence of a defined intracellular target(s), the operation of the
``PtdIns 3-kinase signaling pathway'' will remain enigmatic.
By their nature, lipid-dependent protein kinases are attractive
candidates as targets for the postulated role of PtdIns-3,4,5-P as a second messenger. Protein kinase C (PKC) isotypes constitute
the major group of such enzymes, but several recent reports have
indicated the existence of an additional class of lipid-activated
protein kinases. These kinases, termed PKC-related kinases (PRKs) are
closely homologous to PKC isotypes in the catalytic domain while
retaining a distinct amino-terminal regulatory domain ((3, 4) see Fig. 6A). Unlike the PKCs which characteristically
retain a cysteine-rich C1 domain responsible for effector binding, the
PRKs do not encode a C1 domain but show two distinct conserved
regulatory domains termed HR1 and HR2(3) . Consistent with the
lack of a C1 domain, it has become clear that while the PRKs resemble
PKCs in being activated by proteolysis, they differ from PKCs in not
being activated by phorbol esters. However, like PKC, various fatty
acids and phospholipids have been shown to activate PRKs in vitro(4, 5, 6) .
Figure 6:
The V/HR2 domain is not
required for activation of PKC-
by PtdIns-4,5-P
and
PtdIns-3,4,5-P
. A, schematic diagram showing PRK1,
PKC-
, and the deletion mutant PKC-
(2-137). Both
PRK1 and PKC-
contain a carboxyl-terminal catalytic domain (open box). The amino-terminal regulatory domain of both PRK1
and PKC-
contain several domains of which the HR2 region of PRK1
and the V
of PKC-
share homology (filled circles in each case). The PKC-
(2-137) mutant lacks this
amino-terminal V
domain. Densely hatched boxes in
PRK1 represent the HR1 (a, b, and c) domain. Loosely hatched boxes represent the C1 domain responsible for diacylglycerol and phorbol
ester binding of PKC-
. B, the role of the V
domain in activation by PtdIns-4,5-P
and
PtdIns-3,4,5-P
was examined. Preparations of PKC-
(left-hand panel) and PKC-
(2-137) (right-hand panel) were assayed for activation by 0 mol % (1), 4 mol % (2) and 16 mol % (3)
PtdIns-4,5-P
and PtdIns-3,4,5-P
presented in
Triton X-100 mixed micelles containing 10 mol% PtdSer or, as a positive
control, 50 µg of PtdSer + 50 ng of TPA (4).
In view of the interest
in PtdIns-3,4,5-P as a second messenger, several studies
have addressed the activation of PKC isotypes by this class of
lipids(7, 8, 9) . However, the results show
little consistency in which of the different PKC isotypes are activated
or in the specificity of different lipids. In this paper we address the
specificity of the lipid-kinase interaction in two ways: firstly, with
respect to the target enzyme, and secondly, with respect to the
phospholipid (head group). In order to do so, members of both
lipid-dependent kinase families, PRK and PKC, were compared for
activation by polyphosphorylated inositol lipids in the detergent mixed
micelle assay. This assay, introduced by Hannun et al.(10) for the study of PKC, allows for specific
modulation/stimulation of PKC by phorbol ester and provides a defined
and readily controlled context for presentation of other types of lipid
to kinases. Our results indicate that there seems to be little
specificity for inositol lipid-kinase interactions in vitro.
Unlike PKC, the activation of PRKs by PtdIns-3,4,5-P and PtdIns-4,5-P
has not been documented. Initial
studies with purified PRK1 were performed with pure lipid vesicles
comprised of phosphatidylserine (PtdSer), phosphatidylinositol
(PtdIns), PtdIns-4,5-P
, or PtdIns-3,4,5-P
(Fig. 1A). When presented as pure lipid vesicles,
each of these lipid types were able to activate PRK1 when myelin basic
protein was used as a substrate. All lipids displayed similar
activation profiles (Fig. 1A).
Figure 1:
Effect of various lipid effectors on
PRK1 activity. A, PRK1 was assayed with various concentrations
of PtdSer (--
), PtdIns
(
--
), PtdIns-4,5-P
(
--
), and PtdIns-3,4,5-P
(
--
) presented as sonicated vesicles. B, as A except in the presence of Triton X-100
micelles as described under ``Experimental
Procedures.''
The
concentration-dependent activation of PRK1 observed for lipid vesicles
of PtdSer, PtdIns, PtdIns-4,5-P, and PtdIns-3,4,5-P
provides limited information on specificity/potency in view of
the need to define the surface concentration. In order to work under
defined conditions, Triton X-100 mixed micelles were
employed(10) . In this assay, PtdIns-4,5-P
and
PtdIns-3,4,5-P
showed similar potency in the activation of
PRK1 (Fig. 1B). PtdSer and PtdIns were still able to
fully activate PRK1 but only at 10-fold higher concentrations when
compared to PtdIns-4,5-P
and PtdIns-3,4,5-P
.
In employing detergent mixed micelles in the study of PKC, it has
been well documented that diacylglycerol-dependent activation of
particular PKC isotypes is readily observed in the context of Triton
X-100/PtdSer mixed micelles(10) . Thus, the effects of
PtdIns-3,4,5-P and PtdIns-4,5-P
were studied
under similar conditions (in a background of Triton X-100 mixed
micelles containing 10 mol % PtdSer). It was found that PtdSer at this
concentration has no significant effect on PRK1 activity (Fig. 1B). The presence of PtdSer at 10 mol % in these
vesicles does not significantly affect the inositol lipid-dependent
activation of PRK1 (Fig. 2).
Figure 2:
Titration of PtdIns-4,5-P and
PtdIns-3,4,5-P
in Triton X-100/PtdSer mixed micelles. PRK1
was assayed with various concentrations of PtdIns-4,5-P
(
--
) and PtdIns-3,4,5-P
(
--
) in a background of 10 mol % PtdSer
and in the context of Triton X-100
micelles.
To compare the inositol lipid
activation of PRK1 with that of representative PKC isotypes, a Triton
X-100/10% PtdSer mixed micelle assay was employed (Fig. 3A). It is evident that at 16 mol %
PtdIns-4,5-P or PtdIns-3,4,5-P
, the phorbol
esterresponsive PKC isotypes were all activated to an extent comparable
with that seen in the presence of TPA. In the case of PKC-
, which
is insensitive to TPA (see Fig. 3A(15) ),
activation was also observed, albeit only about 2-fold above basal
activity. Consistent with the previous data ( Fig. 1and Fig. 2), PRK1 also showed a robust activation at 16 mol %
PtdIns-4,5-P
or PtdIns-3,4,5-P
.
Figure 3:
Activation of PRK1 and PKCs by
PtdIns-4,5-P and PtdIns-3,4,5-P
in Triton X-100
micelles. A, PRK1 and various PKCs were assayed in the
presence of 10 mol % PtdSer (1), 10 mol % PtdSer + 4 mol
% PtdIns-4,5-P
(2), 10 mol % PtdSer + 16 mol
% PtdIns-4,5-P
(3), 10 mol % PtdSer + 4 mol %
PtdIns-3,4,5-P
(4), 10 mol % PtdSer + 16 mol
% PtdIns-3,4,5-P
(5), and 50 µg of PtdSer
+ 50 ng of TPA (6). Assays were carried out in the
presence of 1.625 mM MgCl
. B, as in A except for the presence of 12.5 mM Mg
(final) and 0.75 mM Ca
(final) in the
assay.
The
activation observed by all the PKCs and PRK1 is extremely sensitive to
the concentration of Mg and Ca
employed in the assay (Fig. 3B). Excess of these
divalent cations negates the activation observed by PtdIns-4,5-P
and PtdIns-3,4,5-P
, further enforcing the idea that
the anionic charge of these lipids is important for activation. For
PRK1, the effect of Mg
on PtdIns-4,5-P
and PtdIns-3,4,5-P
activation is shown in Fig. 4. The effect of Mg
is clearly biphasic,
being optimum at physiological Mg
concentrations
(
1 mM). The observation that activation by
PtdIns-3,4,5-P
is more sensitive to inhibition by high
concentrations of Mg
is consistent with the notion
that the lipid effects are charge-dependent. This idea is further
supported by the observation that at high concentrations the inositol
polyphosphate InsP
and the related synthetic sulfated
compound InsS
are also able to cause activation of PRK1 in
the 20-100 µM range (Fig. 5).
Figure 4:
Activation of PRK1 by PtdIns-4,5-P and PtdIns-3,4,5-P
is sensitive to
[Mg
]. The effect of titrating
[Mg
] on the activity of PRK1 was measured.
Duplicate assays were carried out in Triton X-100 micelles containing
either 16 mol % PtdIns-4,5-P
(
--
), 16
mol % PtdIns-3,4,5-P
(
--
), or no
effector (
--
).
Figure 5:
Effect of InsP,
InsP
, and InsS
on PRK1 activity. A,
PRK1 was assayed with various concentrations of the inositol
polyphosphates InsP
(
--
) and
InsP
(
--
) and the inositol
polysulfate InsS
(
--
) in the absence
of detergent. B, as A except in the presence of
Triton X-100 micelles as described under ``Experimental
Procedures.''
While there
is a lack of distinction in comparing PRK1 and the phorbol
ester-responsive PKC isotypes, there is a domain in PRK1 (HR2) that is
related to the V domains of PKC-
and -
(see Fig. 6A). In order to test whether this domain
contributed to the inositol lipid activation observed, a deletion
mutant of PKC-
(Fig. 6A(13) ) was
expressed and partially purified. Comparison of PKC-
with
PKC-
(2-137) showed that there was no difference in
lipid activation (Fig. 6B). The apparent nonselectivity
of protein kinase activation in vitro by these inositol lipids
was further studied in the context of mixed brain phospholipids. As
shown in Fig. 7for PKC-
, PKC-
,
PKC-
, and PRK1, there is little distinction in either potency or
efficacy for PtdIns-3,4,5-P
and PtdIns-4,5-P
with any one of these kinases.
Figure 7:
Activation of PRK1, PKC-,
PKC-
and PKC-
by PtdIns-4,5-P
and
PtdIns-3,4,5-P
in Triton X-100 micelles containing 10 mol %
mixed brain phospholipids. The effect of titrating PtdIns-4,5-P
(
--
) and PtdIns-3,4,5-P
(
--
) was measured on the activity of
PRK1, PKC-
, PKC-
, and PKC-
in 10 mol %
mixed brain phospholipid/Triton X-100
micelles.
The data presented here indicate that PRK1 can be activated
by pure lipid vesicles of PtdIns-4,5-P and
PtdIns-3,4,5-P
, to an extent comparable with vesicles of
PtdSer and PtdIns. This is consistent with the activation of PRK1 by a
number of different phospholipids(4, 5, 6) .
Activation of PRK1 by these pure lipid vesicles may or may not reflect
a true activator role. In the case of PKC, PtdSer is a potent activator
when presented alone; however, in detergent (or mixed phospholipid)
micelles, activation of PKC becomes dependent upon diacylglycerol or
phorbol esters(10) . In view of the wealth of literature on the
(physiological) activation of PKC by diacylglycerol or phorbol esters
in intact cells, it can be surmised that the physical status of the
pure phospholipid vesicles poorly reflects the situation in
vivo. By contrast, while not physiological, the detergent mixed
micelles support an in vitro behavior of PKC in keeping with
that observed in vivo.
Employing the Triton X-100 mixed
micelle assay revealed that PtdSer and PtdIns activation of PRK1 was
not achieved until 10-fold higher effector concentrations were employed
compared with PtdIns-4,5-P and PtdIns-3,4,5-P
.
A similar effect, comparable with that seen with PtdSer and PtdIns, was
also observed for mixed brain phospholipids(5) . The dependence
and extent of PRK1 activation was indistinguishable for the two
polyphosphoinositides (PtdIns-4,5-P
and
PtdIns-3,4,5-P
) tested; given the uniform context in which
they are presented (i.e. Triton X-100), this similarity is
likely to reflect a similar mode of action. In principle, there may be
some absolute nonspecific requirement for phospholipid in order to
observe selective activation by one of these phosphoinositides.
However, similar activation profiles were observed for PRK1 whether
Triton X-100, Triton X-100/PtdSer, or Triton X-100/mixed brain
phospholipids were employed. In each case, the A
values for the polyphosphoinositides were between 4 and 6 mol %.
This represents
7 molecules of phosphoinositide per Triton X-100
micelle (140 molecules total(10) ). As a point of reference,
1-2 mol % diacylglycerol (i.e. 2-3
molecules/micelle) is required for PKC activation under similar
conditions(10) .
In parallel to assessing the behavior of
PRK1, various PKC isotypes were also analyzed. With respect to the
polyphosphoinositides, the PKC isotypes studied (aside from PKC-)
behaved essentially as PRK1. Activation of PKC-
,
PKC-
, and PKC-
was observed for both PtdIns-4,5-P
and PtdIns-3,4,5-P
. Furthermore, the extent of
activation was comparable with that observed for optimal Triton
X-100/PtdSer/TPA concentrations. Once again, as with PRK1, no
specificity was observed between these two polyphosphoinositides, each
showing similar A
values in both Triton
X-100/PtdSer and Triton X-100/mixed brain phospholipids. This lack of
distinction between PKC isotypes and between these two lipids is at
variance with some previously published
data(7, 8, 9) . However, these studies have
not assessed activation in the context of detergent mixed micelles and,
as such, variations between phosphoinositides may have reflected in
part alterations to the vesicle structures, these considerations have
been discussed extensively for PKC activation (see, for example, (16) ). Furthermore, there appears to be a significant
variation in regulatory properties of PKC isotypes that is a function
of source, purity, and storage which may vary in the different studies.
For example, we have found this to be a particularly acute problem for
PKC-
which when expressed in Sf9 or Hi5 insect cells progressively
loses its lipid/TPA dependence on purification and storage (discussed
in (17) ).
The modest activation of PKC- under the
conditions tested here (
2-fold) is similar to the extent of
activation we have observed for PKC-
when pure PtdSer vesicles are
employed (data not shown). Thus, in a sense, PKC-
behaves as all
the other kinases studied here, it is only the absolute extent of
activation above basal that varies. The variation with the
responsiveness (9) and nonresponsiveness (8) described
by others may reflect distinct basal activities from the different
enzyme sources.
The similar behavior of all these lipid-activated
kinases toward the two polyphosphoinositides tested suggests that the
interaction in vitro may be nonphysiological since: (i)
PtdIns-3,4,5-P would itself be derived from
PtdIns-4,5-P
and yet both show a similar potency in these
assays, i.e. the activation status of the putative target
would not change and (ii) there is no conservation of potential ligand
binding sites when comparing the PKC family with that of the PRKs. The
latter point is worthy of further comment since there is similarity
between the PKC-
/
V
domain and that of the
PRK1/2 HR2 domain(3) . In order to address the idea that this
domain may be of importance, we tested a V
deletion mutant
of PKC-
((13) see Fig. 6) and observed that it displays
the same polyphosphoinositide response as wild-type PKC-
. This
lack of an identifiable, conserved binding site is consistent with the
lack of distinction between these polyphosphoinositides and suggests
that the interaction reflects a more general anionic phospholipid
requirement of the respective lipid binding domains of these kinases.
The activation observed for all the PKCs and PRK1 is extremely
sensitive to the concentration of Mg
and
Ca
employed in the assay and the observation that at
high concentrations the inositol polyphosphate InsP
and the
related InsS
are able to cause activation of PRK1 (Fig. 5) further enforce the idea that the charge effects in
these activations are important. The fact that the polyphosphoinositide
(PtdIns-3,4,5-P
and PtdIns-4,5-P
) effects are
able to activate within the 4-8 mol % range in Triton X-100
indicates that there is some property of these lipids that promotes
their interaction when compared with PtdSer and PtdIns, the clustered
phosphate groups being the obvious candidates.
In conclusion, PRK1
and members of the PKC family are activated with a similar potency by
PtdIns-4,5-P and PtdIns-3,4,5-P
when presented
in a uniform detergent mixed micelle context. While these lipids might
share a common specific binding site, no such discernible site is
obvious when comparing domains of PRK1 with those of the PKC isotypes.
The implication is that if either lipid is involved in the specific
activation of either class of protein kinases in vivo then
other cofactors and/or distinct conditions pertain. This issue will
only be resolved in a physiological context.