From the Vollum Institute, Portland, Oregon 97201
Specificity is perhaps the most enigmatic
property of hormone-mediated signaling pathways, especially when one
considers that more than 30 hormones employ the ubiquitous second
messengers, Ca2+, phospholipid, or cAMP, to relay messages
from the cell membrane to intracellular effectors. In most cases the
net signaling effect is to activate protein kinases or phosphoprotein
phosphatases, which, in turn, alter the phosphorylation state of
cellular target proteins. Although both enzyme classes have been
intensely researched since the late 1950s, it is still unclear how
individual hormones activate the correct pool of kinase or phosphatase
to trigger specific intracellular events. One explanation that has
recently attracted attention is a "targeting hypothesis" proposing
that phosphorylation events are controlled in part by where kinases and
phosphatases are located in the cell (1). In accordance with this
proposal, it has become apparent that intracellular targeting of both
enzyme classes is determined by association with "targeting
subunits" or "anchoring proteins." While there have been several
general reviews on this subject (1-3), this article will focus on
subcellular targeting of the cAMP-dependent protein
kinase (PKA).1
Regulation and Compartmentalization of the
cAMP-dependent Protein Kinase The primary action of cAMP in eukaryotic cells is to activate PKA
(4). The PKA holoenzyme is a heterotetramer composed of a regulatory
(R) subunit dimer that maintains two catalytic (C) subunits in a
dormant state. Holoenzyme dissociation ensues upon binding of cAMP to
tandem sites in each R subunit. This alleviates an autoinhibitory
contact that releases the active C subunit. The active kinase is then
free to phosphorylate substrates on serine or threonine residues, which
are presented in a sequence context of Arg-Arg-Xaa-Ser/Thr or
Lys-Arg-Xaa-Xaa-Ser/Thr. Given the frequent occurrence of these
sequence motifs in many proteins, it is obvious that unrestricted
access of the C subunit to its substrates would lead to indiscriminate
phosphorylation. Consequently, several regulatory mechanisms are in
place to ensure that cAMP levels and kinase activity are tightly
controlled. Total cAMP levels are determined by a balance of cellular
adenylyl cyclase and phosphodiesterase activities (5, 6), and
signal-terminating mechanisms such as desensitization of adenylyl
cyclase or compartmentalized activation of phosphodiesterases ensure
further localized reduction of the second messenger (7, 8). Although
access to cAMP is the primary requirement for PKA activation,
additional factors are responsible for returning the kinase to the
inactive state. The R subunits are expressed in excess over C subunits
favoring rapid reformation of the holoenzyme when cAMP levels return to the basal state (9). In addition, the ubiquitous heat-stable inhibitor,
PKI, may well serve as a fail-safe device, which sequesters free C
subunit and mediates export of the kinase from the nucleus, which is
devoid of R subunits (10).
For some time now it has been thought that the cellular specificity of
PKA signaling is related to the existence of multiple C and R subunit
isoforms. In mammals, three C subunit isoforms ( The first RII-binding proteins were identified as contaminating
proteins, which co-purified with RII after affinity chromatography on
cAMP-Sepharose. However, a detailed study of these proteins was made
possible by the observation that many, if not all, of these associated
proteins retain their ability to bind RII after they have been
immobilized on nitrocellulose (15). As a result, the standard technique
for detecting RII-binding proteins is an overlay method that is
essentially a modification of the Western blot (reviewed by Carr and
Scott (16)). Using this technique, RII-binding bands ranging in size
from 15 to 300 kDa have been detected in a variety of tissues, and it
would appear that a typical cell contains 5-10 distinct binding
proteins (17). The RII overlay method has also been refined into an
efficient interaction cloning strategy wherein cDNA expression
libraries are screened using RII as a probe. This has led to the
cloning of numerous RII-binding proteins (17-26). More recently, the
RII-binding proteins were renamed A-kinase
anchoring proteins or AKAPs to account for
their proposed PKA-targeting function (26). A model is presented in Fig. 1, which illustrates the essential features of
AKAPs. Each anchoring protein contains two classes of binding sites: a
conserved "anchoring motif," which binds the R subunit of PKA and a
"targeting domain," which directs the subcellular localization of
the PKA-AKAP complex through association with structural proteins,
membranes, or cellular organelles.
Early work in the field focused on mapping the sites on RII
required for interaction with AKAPs. Initially, a family of deletion mutants and chimeric proteins was screened by the overlay to define the
minimum region of RII required to bind MAP2 and AKAP75 (27). It was
concluded that RII dimerization was a prerequisite for anchoring, and
AKAP binding required the first 30 residues of RII (27-29). However,
the localization and dimerization determinants were later shown to be
distinct as deletion of residues 1-5 abolished the anchoring but had
no qualitative effect upon dimerization (29). This led to the
identification of isoleucines at positions 3 and 5 as essential
determinants for association with AKAPs (29, 30). Since leucines and
isoleucines are also crucial determinants of the reciprocal binding
surface on the AKAP (31) it is possible that RII/AKAP docking may be
analogous to the hydrophobic interactions that maintain a leucine
zipper in transcription factors such as CEBP and CREB. However, it must
be noted that the protein-protein interactions required for RII-AKAP
interaction are more elaborate and involve three polypeptide chains,
i.e. two RII protomers and a binding surface on the AKAP
(Fig. 1). Furthermore, additional AKAP binding determinants have been
mapped between residues 11 and 25 of each RII molecule (32).
An added level of specificity in PKA signaling may be achieved through
the differential localization of RII Complementary studies have mapped the corresponding site on AKAPs that
binds the RII dimer. Deletion analyses located the RII-binding
sequences of MAP2 and AKAP150, the murine homologue of AKAP75, to short
continuous regions of amino acid sequences (35, 36). However, the
nature of the RII-binding motif remained unclear until a human
thyroid-anchoring protein called Ht31 was identified (17, 37). The
RII-binding sequence of Ht31 exhibited sequence similarities to both
MAP2 and AKAP150 and was predicted to form an Functional Consequences of PKA Anchoring Knowledge of the RII-binding domains on several AKAPs has allowed
the generation of reagents that alter PKA anchoring within cells. So
far two experimental strategies have been used: overexpression of AKAPs
or RII-binding fragments (Fig. 2B) and the
introduction of peptides that disrupt anchoring. Overexpression of
AKAP75 or its human homolog AKAP79 redirects RII and other enzymes to
the periphery of HEK 293 cells (38, 39), whereas expression of an
untargeted RII-binding fragment called AKAP45 prevents membrane targeting of RII
Peptides encompassing the amphipathic helix region of Ht31 (residues
493-515) effectively compete for RII-AKAP interaction in
vitro and disrupt the subcellular localization of PKA inside cells
(41). Perfusion of cultured hippocampal neurons with these "anchoring
inhibitor peptides" caused a time-dependent decrease in
AMPA/kainate-responsive currents, whereas perfusion of control peptides, which were unable to compete for RII binding, had no effect
on channel activity (41). Additional controls confirmed that the
effects emanated from PKA as perfusion of PKI peptides, which block
kinase activity, caused a decrease in channel activity, whereas
microinjection of excess C subunit overcame the anchoring inhibitor
effect. Collectively, these findings suggested that the Ht31 peptide
displaced PKA from anchored sites close to the AMPA/kainate channels,
thereby decreasing the probability of channel phosphorylation. Parallel
studies by Catterall and colleagues have subsequently shown that Ht31
peptide-mediated disruption of PKA anchoring modulates L-type
Ca2+ channels in skeletal muscle (42). Recently, this group
has postulated that the peptides may uncouple the association of RII with a low molecular weight AKAP (AKAP15) that co-purifies with the
channel (43). Taken together, these studies provide convincing evidence
that PKA anchoring may facilitate preferential modulation of
physiological PKA substrates.
However, there are technical limitations associated with the
introduction of bioactive peptides into cells. Although microinjection or microdialysis is suitable for peptide delivery into single cells,
the uptake of lipid-soluble peptide analogues is necessary to affect
many cells. Accordingly cell-permeant anchoring inhibitor peptides have
been developed. Myristoylated Ht31 peptides have been used to
demonstrate a role for PKA anchoring in the cAMP-induced attenuation of
interleukin-2 transcription in Jurkat T
cells,2 and stearoylated Ht31 analogues
have been shown to arrest the motility of mammalian sperm (44). An
intriguing aspect of the latter study is that inhibition of the C
subunit with PKI peptides does not mimic the "anchoring inhibitor
peptide" effect. This has led to the proposal that the R subunit has
a distinct function in the regulation of sperm motility that is
independent of the C subunit (44).
The PKA-anchoring model (Fig. 1) implies that AKAPs must contain a
unique targeting site that is responsible for association with
subcellular structures. The targeting domain is an essential feature of
each AKAP as it confers specificity by tethering the anchored PKA
complex to particular organelles. So far, immunochemical and
subcellular fractionation techniques have identified AKAPs localized to
centrosomes (AKAP350), the actin cytoskeleton (Ezrin/AKAP78, AKAP250,
and AKAP75/79/150), the endoplasmic reticulum (AKAP100), the Golgi
(AKAP85), microtubules (MAP2), mitochondria (sAKAP84), the nuclear
matrix (AKAP95), the plasma membrane (AKAP15), and peroxisomes
(AKAP220). The subcellular locations of these known AKAPs are indicated
in Fig. 2A. Less characterized anchoring proteins have been
identified in secretory granules, plasma membranes, and the flagella of
mammalian sperm (3).
Although an increasing number of compartment-specific AKAPs are now
identified, targeting sequences have been extensively mapped for only
two proteins. A C-terminal octadecapeptide repeat sequence targets the
microtubule-associated protein MAP2 to microtubules (45), whereas two
non-contiguous N-terminal basic regions, called T1 and T2, facilitate
submembrane attachment of AKAP75/79 to the cortical cytoskeleton in HEK
293 cells (38). At this time the extent of AKAP75/79 targeting
interactions is not clear as the T1 region has been independently
identified as a site of contact with another signaling enzyme, protein
kinase C (PKC) (39). This raises the intriguing possibility that AKAP79
could be targeted, in part, through protein-lipid interactions between
PKC and the plasma membrane. However, AKAP79 targeting could also
involve a third signaling enzyme as the phosphatase 2B (PP-2B),
calcineurin, also associates with AKAP79 (46). Thus, submembrane
targeting of AKAP79 could be mediated partly through the myristoyl
moiety on the B subunit of the PP-2B holoenzyme. Conceivably, the T1 and T2 regions may represent two distinct targeting signals since AKAP79 is localized to both the cell bodies and dendrites in neurons (39).
Three other AKAPs appear to have distinct targeting sequences but have
been not been analyzed in depth. AKAP220 may be targeted to peroxisomes
(22) as the last 3 residues of the protein, Cys-Arg-Leu, conform to a
peroxisomal targeting signal 1 motif, which is thought to facilitate
the attachment of proteins to the lipid matrix of the peroxisome (47).
AKAP250 is a component of the membrane/cytoskeleton, which is enriched
in the filopodia of adherent human erythroleukemia cells (24). The N
terminus of AKAP250 contains a consensus myristoylation signal, as well
as other structural regions that bear some resemblance to actin-binding
proteins such as MARCKS and GAP-43 (48). Likewise, an anchoring protein
previously called AKAP78 has now been identified as the cytoskeletal
component ezrin (49). In fact, ezrin and its two close relatives,
radaxin and moesin, bind RII in the overlay assay. All three proteins
(ezrin, radaxin, moesin) are members of the band 4.1 superfamily of
proteins, which link the membrane and the cytoskeleton. Therefore,
cross-linking from the membrane to the cytoskeleton might be a shared
function of anchoring proteins such as AKAP79, AKAP250, and ezrin,
radaxin, moesin (Fig. 2A).
Although the principal function of the AKAPs is undoubtedly to
target PKA, a fascinating new aspect has been the discovery of
anchoring proteins, which simultaneously bind more than one signaling
enzyme. It is thought that these multivalent AKAPs serve as scaffolds
for the assembly of signaling complexes consisting of several kinases
and phosphatases (50). This is an appealing variation on the anchoring
theme as it provides a model for reversible phosphorylation in which
the opposing effects of kinase and phosphatase action are co-localized
in a multiprotein transduction complex (3, 51).
In neurons AKAP79 targets PKA to postsynaptic densities,
cytoskeletal-like dendritic structures located on the internal face of
excitatory synapses. Biochemical studies have shown that AKAP79 also
binds PP-2B (46) and PKC (39). The structure of AKAP79 is modular as
peptide studies and co-precipitation techniques have demonstrated that
each enzyme binds to a distinct region of the anchoring protein. This
has led to the proposal that AKAP79 coordinates the location of three
signaling enzymes at postsynaptic sites (Fig.
3A). Potential substrates for this signaling
complex are likely to be postsynaptic AMPA/kainate receptors or
Ca2+ channels, which can be modulated by AKAP-targeted PKA
(41), and N-methyl-D-aspartate receptors, which
are activated by PKA or PKC and attenuated by PP-2B. A potential role
for AKAP79 in the coordination of synaptic signaling is similar to the
function of sterile 5 (STE5), a yeast scaffold protein that organizes
three protein kinases that govern the pheromone mating pathway (52). There are, however, important differences between these two signaling scaffolds; AKAP79 dictates the subcellular location of two protein kinases of broad specificity and a phosphatase, whereas STE5 organizes the localization and sequential activation of a MAP kinase cascade. Another distinction is that the AKAP79 scaffold integrates signals from
three second messengers, cAMP, Ca2+/phospholipid, and
Ca2+/calmodulin, which activate three independent enzymes.
In contrast, a single upstream event, the activation of STE20, is
sufficient to transduce a signal from one kinase to the next in the
STE5 signaling scaffold. Nevertheless, the similarities between AKAP79 and STE5 provide an opportunity to speculate that other anchoring proteins may also function as signaling scaffolds.
AKAP250, the anchoring protein enriched in the filopodia of adherent
megakaryocytes (Fig. 2B), also contains binding sites for
both RII and PKC (24). It was first noticed that the C-terminal third
of AKAP250 was identical in sequence to that of a previously cloned
protein called gravin, which was identified as a cytoplasmic antigen
recognized by sera from patients with myasthenia gravis (53).
Gravin/AKAP250 is depicted in Fig. 3B as a scaffold linking an actin target site and the cell membrane. When the complete amino
acid sequence of gravin/AKAP250 was available it became apparent that
distinct regions were similar to other AKAPs and to a PKC
substrate/binding protein called SSECKs or clone 72 (54, 55). The
PKA-binding site on gravin was mapped to a region of sequence in which
10 of 14 residues are identical to those in the corresponding region of
AKAP79. This RII-binding sequence is also present in SSECKs/clone 72. The similarity of all three sequences was rather surprising as it had
been proposed that a lack of sequence identity among AKAPs is due to a
conservation of secondary, rather than primary, structure in the
RII-binding motif (14). Therefore, gravin, SSECKs/clone 72, and AKAP79
may be members of a structurally related subfamily of anchoring
proteins, each of which bind more than one kinase or phosphatase and
are targeted to the membrane cytoskeleton.
The cloning of additional AKAPs should provide a more complete
data base of sequences. This information should establish whether the
AKAPs merely represent a convergent group of proteins sharing a common
RII-binding motif or whether there are families of anchoring protein
genes. Although most of the current data supports the first view, it is
noteworthy that sAKAP84 is expressed in several forms due to
alternative splicing; one splice variant, dAKAP1, was isolated in a
two-hybrid screen using an RI Despite the finding that both PKA holoenzyme subtypes may associate
with AKAPs, it remains unclear why a significant fraction of PKA in
most cell types is soluble. When this observation is considered
together with evidence that the number of available anchoring sites in
cells is in excess over the kinase, it seems likely that R-AKAP
interactions are regulated in some manner. Although phosphorylation of
RII
,
, and
)
exist, and although there are subtle differences in the kinetic
characteristics and cAMP sensitivities of C
- and C
-containing
holoenzymes, these two predominant C subunit isoforms are virtually
indistinguishable with respect to substrate specificity and interaction
with R subunits (4, 11). In contrast, the dimeric R subunits exhibit
both distinct cAMP binding affinities and differential localization
within cells (4, 12). The type I PKA holoenzyme (containing either
RI
or RI
) is predominantly cytoplasmic, whereas >75% of the
type II PKA holoenzyme is targeted to certain intracellular sites
through association of the RII subunits (RII
or RII
) with
cellular binding proteins known as anchoring proteins (previously
reviewed by Rubin (13) and Scott and McCartney (14)). Thus, it has
recently been proposed that differences in subcellular targeting of
type I and type II PKA are additional factors contributing to
specificity in cellular responses.
Fig. 1.
A PKA-anchoring model is depicted.
Binding surfaces on the AKAP for association with PKA
(Anchoring) and for interaction with subcellular organelles
or structures (Targeting) are indicated.
[View Larger Version of this Image (52K GIF file)]
or RII
by association with
isoform-selective AKAPs. Several years ago, it was reported that RII
had a 6-fold preference for MAP2, whereas RII
had a 2-fold
preference for AKAP75 (33). Recently it has been shown that
follicle-stimulating hormone treatment of rat granulosa cells induces
an 80-kDa RII
-selective AKAP (34). Sequences of the first 10 amino
acids of RII
and RII
are almost identical except for a pair of
prolines at positions 6 and 7 in RII
that is not present in RII
.
Removal of this proline pair impairs preferential interaction with
RII
-selective AKAPs (30). Although a precise structural explanation
is not available for these observations, proline 6 may increase RII
affinity for certain AKAPs through direct contact with the anchoring
proteins, or alternatively, the greater rigidity of the imino peptide
linkage may precisely orient other AKAP binding determinants.
Nevertheless, it is clear that the orientation of the RII dimer
(parallel or antiparallel) is another essential component of the
AKAP-binding site as it controls the spatial geometry of isoleucines 3 and 5. Undoubtedly, the completion of studies to solve the structure of
an RII fragment complexed with an AKAP peptide will resolve these
issues.
-helix. Helical wheel
projections of all three sequences exhibited a striking segregation of
hydrophobic and hydrophilic side chains. This led to the proposal that
the RII-binding motif of Ht31 and other AKAPs involves an amphipathic
helix (37). Subsequent studies performed on Ht31 and AKAP79, the human
homolog of AKAPs 75 and 150, demonstrated a requirement for this region in RII binding (19, 37). The role of helical secondary structure in
RII-AKAP interactions was supported by demonstrating that substitution of proline, a residue that perturbs helix formation, at various positions within the RII-binding domain abolished RII binding (19, 37).
These findings were consolidated by the synthesis of peptides
encompassing the predicted helical region of Ht31, which were shown to
bind either RII or the type II PKA holoenzyme with nanomolar affinity
(17). Although the involvement of an amphipathic helix has not been
definitively proven, independent studies have confirmed that similar
regions of several other AKAPs are essential determinants for RII
binding (20, 23, 24). The high affinity of these interactions has
important consequences for the intracellular localization of PKA.
First, the KD for the RII-AKAP interaction has been
calculated from 1 to 11 nM by a variety of analytical
methods. This affinity constant is well within the intracellular
concentration ranges of RII and most AKAPs suggesting that the RII-AKAP
complex will be favored in situ. Second, the PKA holoenzyme
binds Ht31 with the same high affinity as the RII dimer. Thus, the PKA
holoenzyme will be anchored in cells when cAMP is at basal levels.
in thyroid-derived cell line FRTL-5 (40). The
functional consequences of these studies are not yet clear although
there is some evidence that expression of the AKAP45 fragment prevents
nuclear accumulation of the C subunit and decreases phosphorylation of
the nuclear transcription factor CREB (40), suggesting a role for
anchored PKA in cAMP-mediated transcriptional regulation.
Fig. 2.
Subcellular targeting of AKAPs. A,
schematic diagram indicating the locations of known AKAPs in a
prototypic cell. See text for details. E.R.M., ezrin,
radaxin, moesin. B, subcellular distribution of AKAPs
expressed in cultured cell lines. Each panel shows
immunofluorescent staining with antisera specific for the indicated
AKAPs. Localization of AKAPs 79, 95, and 100 and an untargeted
RII-binding fragment of Ht31 was visualized in transiently transfected
HEK 293 cells. DNA in the nucleus was detected by Hoechst staining, and
gravin expression was detected in human erythroleukemia cells treated
with phorbol esters for 18 h.
[View Larger Version of this Image (99K GIF file)]
Fig. 3.
AKAP signaling complexes. Models are
shown for the AKAP79 signaling scaffold (A) and the proposed
gravin (AKAP250) signaling scaffold (B). The enzymes and
second messenger systems coordinated by these AKAP complexes are shown.
The potential subcellular targeting functions of protein-lipid
interactions involving PKC, PP-2B, or N-terminal myristoylation and
protein-protein interactions involving cytoskeletal components are also
shown for AKAP79 and gravin.
[View Larger Version of this Image (46K GIF file)]
fragment as bait (56). Independent
studies have shown that AKAP79 also binds RI
, albeit with a 100-fold
lower affinity than it does RII
.2 This raises the
intriguing possibility that certain AKAPs are dual function anchoring
proteins, which bind both RI and RII. Not only do these findings
provide a molecular mechanism for the compartmentalization of RI (57),
but they also significantly expand the original anchoring hypothesis to
include all PKA holoenzymes. Undoubtedly, future research will
elucidate the nature of the RI-AKAP interactions and establish the
physiological significance of type I PKA anchoring.
by CDC2 kinase prevents its association with MAP2, this does not
occur with RII
or RI (58). No doubt, future studies will focus on
regulation of PKA anchoring. Another area of future emphasis will be
the characterization of AKAP-targeting domains. AKAPs such as ezrin,
radaxin, moesin, AKAP79, and gravin appear to be anchoring proteins
that are linked to both the plasma membrane and the actin cytoskeleton.
This suggests that highly localized PKA phosphorylation events may
regulate cell shape and motility. Finally, it is likely that additional
AKAP multienzyme signaling complexes, analogous to those that include
AKAP79 and gravin, will be identified. For example, the Tau protein
associates with PP-2A, and it is likely that MAP2, which is very
similar to Tau and an AKAP, will bind PP-2A as well (59). It is also conceivable that AKAP transduction complexes could contribute to the
specificity of cAMP action by bringing PKA together not only with
phosphatases but also with the cAMP phosphodiesterases responsible for
signal termination. These types of macromolecular organization mediated
by anchoring proteins would not only place PKA close to certain
substrates but also cluster the kinase with enzymes that regulate its
activation state and enzymes that control the dephosphorylation state
of the substrate. The challenge now is to pinpoint which important
cellular phosphorylation events are regulated by the anchored kinases
and phosphatases.
We thank our colleagues in the Vollum Institute who critically evaluated this article. We especially thank Drs. Susan Taylor, Patricia Jennings, James Goldenring, Brian Murphy, Daniel Carr, Monique Howard, W. Michael Gallatin, and G. Stanley McKnight for communicating results prior to their publication.
REFERENCES3