From the Department of Molecular Pharmacology, Atran Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, October 2, 2000, and in revised form, December 20, 2000
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ABSTRACT |
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Association of an atypical protein kinase C
(aPKC) with an adapter protein can affect the location, activity,
substrate specificity, and physiological role of the
phosphotransferase. Knowledge of mechanisms that govern formation and
intracellular targeting of aPKC·adapter protein complexes is limited.
Caenorhabditis elegans protein kinase C adapter proteins
(CKA1 and CKA1S) bind and target aPKCs and provide prototypes for
mechanistic analysis. CKA1 binds an aPKC (PKC3) via a
phosphotyrosine binding (PTB) domain. A distinct, Arg/Lys-rich
N-terminal region targets CKA1 to the cell periphery. We discovered
that a short segment (212GGIDNGAFHEHEI224) of
the V2 (linker) region of PKC3 creates a binding surface that interacts with the PTB domain of CKA1/CKA1S. The docking domain of
PKC3 differs from classical PTB ligands by the absence of Tyr and Pro.
Substitution of Ile214, Asn216, or
Phe219 with Ala abrogates binding of PKC3 with CKA1; these
residues cooperatively configure a docking site that complements an
apolar surface of the CKA1 PTB domain. Phosphorylation site domains
(PSD1, residues 11-25; PSD2, residues 61-77) in CKA1 route the
adapter (and tethered PKC3) to the cell periphery. Phosphorylation of Ser17 and Ser65 in PSDs 1 and 2 elicits
translocation of CKA1 from the cell surface to cytoplasm. Activities of
DAG-stimulated PKCs and opposing protein Ser/Thr phosphatases can
dynamically regulate the distribution of adapter protein between the
cell periphery and cytoplasm.
Atypical protein kinase C
(aPKC)1 isoforms, which
include mammalian PKCs The nematode C. elegans is an attractive model system for
studies on aPKC adapter proteins. C. elegans physiology is
regulated by signaling molecules, mechanisms and pathways that are
operative in mammals (21, 22). Only one aPKC (PKC3) is encoded by the C. elegans genome (3). PKC3 is expressed and anchored at all developmental stages (3). This Ca2+- and diacylglycerol
(DAG)-independent kinase is essential for the progression of
embryogenesis, asymmetry in early cell divisions, and overall viability
of the organism (3, 11). In 1-cell embryos, ~25% of PKC3 associates
with Par-3, a multi-PDZ domain protein that is crucial for
generating intracellular polarity (11, 12). Mechanisms governing
formation of PKC3·Par-3 complexes, the identity of targets for
bound PKC3, and the precise biochemical/physiological function of the
complex remain to be defined. Association of >90% of PKC3 with
cytoskeleton/membranes in embryos indicates that additional adapter
proteins are expressed during early phases of C. elegans
development (3). In post-embryonic C. elegans, PKC3
accumulates in a highly asymmetric fashion in intestinal, pharyngeal,
and other cells (3). Polarized enrichment of PKC3 in nondividing cells
that will not undergo apoptosis suggests that anchored PKC3 plays
distinct roles in terminally differentiated cells. By using knowledge
of the established properties of C. elegans PKC3 and
reagents derived from PKC3 cDNA and protein (3), it should be
possible to discover and characterize a cohort of adapter proteins that
collectively diversify aPKC functions from birth to death.
A yeast two-hybrid interaction screen yielded a unique C. elegans cDNA that encodes two novel PKC3-binding proteins
(45). These proteins, which were named protein kinase C adapter 1 (CKA1) and CKA1S, are expressed throughout the life span of C. elegans and accumulate near the inner surface of plasma membranes
in vivo and in transfected cells (45). In highly polarized
epithelial cells, CKA1 and CKA1S are differentially targeted to the
lateral plasma membrane surface (near tight junctions). CKA1 (593 amino acids) contains a positively charged N-terminal region (residues 1-89)
that precedes a phosphotyrosine binding (PTB) domain (residues 90-231)
and a unique central/C-terminal segment (residues 232-593). The
minimal fragment of CKA1 polypeptide that tethers PKC3 corresponds to
the intact PTB domain (45). Deletion mutagenesis-transfection experiments indicate that the N-terminal region of CKA1 governs routing
of the PKC3 adapters to the cell periphery (45). Two distinct,
exceptionally basic clusters of amino acids (residues 11-25 and
residues 61-77) seem to play important roles in targeting and
anchoring CKA1. The composition and arrangement of amino acids within
the clusters generate sequence motifs that resemble classical phosphorylation site domains (PSDs) of the ubiquitous MARCKS protein and MARCKS-related proteins (29, 30). Utilization of an alternative initiation codon in CKA1 mRNA results in the synthesis of CKA1S (45). The amino acid sequence of this adapter isoform is identical with
residues 45-593 in CKA1. Therefore, CKA1S lacks the first PSD but
retains all other structural and functional features of CKA1. Our
current model proposes that a PTB domain and PSDs in CKA1/CKA1S
collaborate in a novel manner to achieve the targeting, tethering, and
functional diversification of an atypical PKC.
Structural and biochemical mechanisms that govern the formation,
destination, and functions of CKA1·PKC3 complexes are not thoroughly
understood. In particular, we lack knowledge of (a) properties of PKC3 that promote interactions with CKA1 and
(b) mechanisms by which the CKA1 N-terminal region
(including PSDs) routes the adapter protein to the cell periphery. To
address these topics, we attempted to answer a series of fundamental
questions. (a) What structural features enable PKC3 to
engage the CKA1/CKA1S PTB domain? (b) Does PKC3 contain a
phosphotyrosine that is essential for formation of a stable complex
with CKA1 or CKA1S? (c) Are the adapter proteins candidate
substrates (or substrate-effectors) for PKC3 and/or DAG-activated PKCs?
(d) Are Ser and Thr residues within the N-terminal regions
of CKA1 and CKA1S phosphorylated? (e) If so, what is the
functional significance of N-terminal phosphorylation? We now report
the results and conclusions from experiments designed to answer the
questions posed above.
Deletion and Site-directed Mutagenesis--
Deletion mutagenesis
was performed on PKC3 cDNA via the polymerase chain reaction, as
previously described (31, 32, 45). For N- or C-terminal deletions of
the regulatory region of PKC3 (nucleotides 1-699), the 5' and 3' ends
of cDNAs encoding desired segments of PKC3 were extended with
NotI and BamHI restriction sites, respectively,
and cloned into the yeast expression vector pAS1. In separate
reactions, SpeI and NotI restriction sites were appended at the 5' and 3' ends, respectively, of cDNAs encoding desired fragments of PKC3. Amplified cDNAs were cloned into the mammalian expression vector pEBG (33). This enabled expression of GST
partial PKC3 fusion proteins in hamster AV-12 cells (see "Results and
Discussion" for details). Amino acid substitutions were introduced
into full-length CKA1 and partial PKC3 polypeptides by PCR-based
site-directed mutagenesis (QuickChangeTM kit, Stratagene).
Mutagenesis reactions were carried out according to the manufacturer's
instructions. All mutants were verified by DNA sequencing.
Cell Culture and Transfections--
Hamster AV-12 cells were
grown as described previously (35, 45). Cells were transfected with GST
partial PKC3 transgenes (inserted in recombinant pEBG vectors) via
calcium phosphate precipitation as reported previously (34, 35). The
same methodology was used to introduce wild type and mutant CKA1 and
CKA1S transgenes (inserted in recombinant pRc/CMV vectors (45)) into
AV-12 cells. Stable transfectants were obtained by selection with 1 mg/ml G418 for 14 days (34).
Precipitation of CKA1/CKA1S from Cell Extracts by GST-PKC3 Fusion
Proteins--
AV-12 cells were transfected with expression plasmids
encoding various GST partial PKC3 fusion proteins. Cells were harvested 24 h after transfection and lysed with 20 mM Tris-HCl,
pH 7.5, containing 0.2 mM dithiothreitol, 0.15 M NaCl, 1 mM EDTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 2 µg/ml pepstatin, 10 µg/ml soybean trypsin
inhibitor, 10 mM benzamidine HCl, 100 µg/ml pefabloc, and
0.5% (w/v) Triton X-100. GST-PKC3 proteins (from one 10-cm plate) were
isolated and purified by incubation with 35 µl of GSH-Sepharose 4B
beads at 4 °C for 1 h. Subsequently, the beads were pelleted
(3,000 × g) and washed twice with 1 ml of lysis buffer. One-third of the beads were mixed with protein (250 µg) extracted from AV-12 cells that were transfected with a CKA1 or CKA1S
transgene. Various concentrations (0.05 µM to 2 mM) of peptides (SGGGIDNGAFHEHEI or AHNIFGISGEHGEDG, see
"Results and Discussion") were included at this step in competition
binding experiments. Samples were incubated at 4 °C for 1 h.
Beads were pelleted at 3,000 × g, and the supernatant
solution was removed. Next, the beads were washed 5 times by suspension
in 1 ml of 10 mM sodium phosphate, pH 7.4, containing 0.15 M NaCl and 0.1% (w/v) Tween 20, and centrifugation at
3,000 × g. Bound proteins were released from the beads
by boiling in 25 µl of SDS gel loading buffer, fractionated by
denaturing electrophoresis, and transferred to an Immobilon P membrane
as described previously (3, 36). Blots were probed with anti-CKA1 IgGs,
and antigen-IgG complexes were visualized by an enhanced
chemiluminescence procedure as reported previously (3, 34).
Phosphorylation of PSD1 and PSD2 by PKC3--
Complementary DNAs
encoding PSD1 (residues 1-63) and PSD2 (residues 59-108) from CKA1
were synthesized via PCR and cloned into the bacterial expression
plasmid pGEX-KG (37). This enabled synthesis in Escherichia
coli of two fusion proteins, GST-PSD1 (designated PSD1') and
GST-PSD2 (PSD2'), that contain potential PKC phosphorylation sites. The
soluble fusion proteins were purified to near-homogeneity by affinity
chromatography as described previously (38). Purified PKC3 (10 ng, Ref.
3) was mixed with various amounts of GST-PSD fusion proteins in 30 µl
of kinase reaction buffer (25 mM Tris-HCl, pH 7.4, 50 µM [ Other Experimental Procedures--
Descriptions of production
and affinity purification of antibodies, yeast two-hybrid protein
interaction assays, denaturing electrophoresis, Western immunoblot
analysis, DNA sequencing, immunoprecipitations, and immunofluorescence
analysis of the intracellular distribution of wild type and mutant CKA1
and CKA1S proteins are provided in the accompanying paper (45).
The CKA1-binding Site in PKC3 Contains Six Core Amino Acids and
Lacks Tyr--
N- and C-terminal boundaries of the segment of PKC3
that engages the PTB domain of CKA1 were mapped by employing deletion mutagenesis in concert with yeast two-hybrid protein interaction assays. Both full-length PKC3 and a large N-terminal fragment of the
kinase (residues 1-233, designated PKC3-N233) avidly bound the PTB
domain (Fig. 1, A and
B). In contrast, a segment of PKC3 that encompasses amino
acids 234-597 was not a PTB ligand (data not shown). Thus, the
catalytic domain (residues 250-515, Ref. 3) and C-terminal region
(residues 516-597) of PKC3 (Fig. 1C) do not mediate binding
with CKA1/CKA1S. Elimination of sequences corresponding to the
pseudosubstrate site (residues 109-121), the Cys-rich regulatory
region (residues 128-177), and/or the unique N-terminal portion
(residues 1-89) of PKC3 failed to disrupt coupling with the CKA1 PTB
domain (Fig. 1A,
Mammalian cells were used to directly and independently test
conclusions drawn from assays performed in the yeast two-hybrid system.
The expression plasmid pEBG contains a multienzyme cloning site that is
preceded by the strong, constitutive elongation factor 1
If residues 212-224 independently generate a binding site for the CKA1
PTB domain within the intact PKC3 protein, then a 15-mer peptide
(designated CBSP for CKA1-Binding
Site Peptide) that contains the same sequence
of amino acids (210SGGGIDNGAFHEHEI224) might
efficiently inhibit formation of PKC3·adapter protein complexes. CBSP
proved to be a potent inhibitor of the tethering of PKC3 by CKA1
(apparent Mr = 75,000) and CKA1S (Fig.
3, A and B).
Binding of PKC3 with the CKA1/CKA1S PTB domain was reduced 90-99% by
50-500 µM CBSP (Fig. 3A). Moreover, 0.5 µM CBSP blocked sequestration of PKC3 by CKA1/CKA1S by
slightly more than 50% relative to a scrambled peptide with the same
amino acid composition as residues 210-224 of PKC3 (Fig.
3B). Thus, the CBSP sequence module in PKC3 appears to be
necessary and sufficient for tethering the kinase to an
adapter-targeting protein.
Site-directed mutagenesis was used to characterize individual amino
acids that govern tethering of PKC3 by CKA1/CKA1S. Key results are
presented in Fig. 4A.
Substitution of either Ile214, Asn216, or
Phe219 with Ala abrogated the ability of PKC3 to bind with
CKA1 and CKA1S. Mutation of other residues (as typified by
His222 to Ala) had no effect on association of PKC3 with
its cognate PTB domain. The data suggest that (a) a
6-residue core region (214IDNGAF219) contains
several amino acids that are critical for ligation of PKC3 by a PTB
domain, (b) large aliphatic, neutral, and aromatic side
chains of Ile214, Asn216, and
Phe219 cooperatively produce a binding surface that
complements (in part) an apolar surface created by Phe175
and Phe221 in CKA1/CKA1S (45), and (c) neither
Pro nor Tyr are essential for the binding of PKC3 with a PTB domain.
The ability of the CBSP module to engage the PTB domain in
vivo was tested in competition assays. CKA1 and CKA1S route
tethered PKC3 to the periphery of transfected AV-12 cells (45).
However, cotransfection with an additional highly expressed transgene
encoding GST partial PKC3 (residues 111-227) protein, elicited
dispersion of the full-length aPKC throughout the cytoplasm (Fig.
4C, panel 1). In contrast, neither GST alone nor
a mutant GST partial PKC3 (residues 111-227) that contains an
Asn216 to Ala substitution affects CKA1-mediated targeting
of intact aPKC to the cell surface (Fig. 4C, panels
2 and 3). Thus Asn216, a critical core
component of the CBSP module (Figs. 3 and 4A), is essential
for competitive docking of fusion protein ligand with
membrane-associated CKA1 in situ. Results in Fig.
4C strongly support the idea that the CBSP region of PKC3
(initially identified by in vitro binding assays) is a
ligand for the CKA1 PTB domain in the context of intact cells.
Recent studies prompted a revision of concepts regarding the binding of
ligands by PTB domains (25-28, 39). The initial postulate that PTB
domains couple with NPXpY motifs (X corresponds
to any amino acid and pY indicates phospho-Tyr) is now recognized as a
specific example of a more general consensus. The majority of PTB
ligands do not contain phospho-Tyr and several PTB domain-partner proteins lack both Pro and Tyr in their binding sites (25-28). Margolis and co-workers (39) proposed a new PTB ligand consensus that
applies in nearly all cases (Fig. 5). A
core region of 6 amino acids is required. Residue 1 is aromatic or has
a large aliphatic side chain; residue 3 is an invariant Asn, and
residue 6 is Tyr or Phe. The PTB ligand site in PKC3 matches perfectly with the optimized consensus motif (Fig. 5). Moreover, an N-terminal Gly residue that is evident in binding partners for
Drosophila Numb, mammalian Numb, and X11 (a
Xenopus PTB-containing adapter) is also shared with the
target (PKC3) for the PTB domain in C. elegans CKA1 (Fig.
5). Amino acids in PKC3 that generate the CKA1 ligation site presumably
fold into a
All other candidate binding/scaffold proteins for aPKCs (discussed by
Zhang et al. (45)) couple with either the catalytic domain
or pseudosubstrate/Cys-rich regions of aPKCs (15-17, 40). In contrast,
our results assign a unique functional role to a region of aPKCs that
was previously presumed to be a structural element. A comparable
PTB-binding site is not evident in the V2 region of
Ca2+ and/or DAG-activated, C. elegans PKCs 1 and
2 (41, 42). This suggests that the PTB-binding site in PKC3 contributes
to both the complexity and specificity of aPKC-mediated signal transduction.
PKC3 Phosphorylates Ser17 and Ser65 in the
PSDs of CKA1--
CKA1 contains two N-terminal PSD-like regions (amino
acids 11-25 and 61-77 in Fig.
6A) that are potential targets
for PKC3-mediated phosphorylation. When CKA1S, which contains only the
second PSD site, was isolated from transfected AV-12 cells, it served
as a good substrate for PKC3 in vitro (Fig. 6B, lanes
3 and 4). However, coexpression of PKC3 and CKA1S
transgenes yielded a CKA1S polypeptide that was a poor substrate for
purified PKC3 (Fig. 6B, lanes 1 and 2). Thus,
CKA1S was evidently phosphorylated by overexpressed PKC3 in the milieu
of intact cells. Inspection of the PSD-1 (amino acids 11-25, Fig.
6A) and PSD-2 (amino acids 61-77, Fig. 6A)
regions disclosed that Ser17 and Ser65 are in
near-optimal sequence contexts for the phosphotransferase activity of
the aPKC. Wild type and mutant GST fusion proteins that include
residues 1-63 (GST-PSD1') and residues 59-108 (GST-PSD2') were
synthesized in E. coli and purified to near-homogeneity by affinity chromatography. Vmax and
Km values for PKC3-catalyzed phosphorylation of wild
type GST-PSD1' and GST PSD2' (Fig. 6, D and E)
were similar to values obtained (Km = 1.3 µM, Vmax = 2.5 pmol/min) for a
PKC3 pseudosubstrate-derived peptide (YRRGSRRWKKIY, which corresponds
to residues 112-123 in PKC3 except for the substitution of
Ala116 with Ser, see Ref. 3). GST alone is not
phosphorylated by PKC3. The kinetic constants indicate that the PSD1
and PSD2 domains are excellent substrates for PKC3. The efficiency of
phosphorylation of these targets may be substantially amplified by the
tethering of the kinase to the PTB domains of intact CKA1 and CKA1S
adapter proteins. Substitution of Ala for Ser17 or
Ser65 abolished the abilities of PSD1' and PSD2',
respectively, to serve as PKC3 substrates (Fig. 6C).
Mutation of other Ser residues in PSD1' and PSD2' (e.g.
Ser76 to Ala, Fig. 6C) failed to diminish
incorporation of 32P radioactivity. Thus, each of the
N-terminal PSDs of CKA1 contains a unique target site for
PKC3-catalyzed phosphorylation.
The Intracellular Distribution of CKA1/CKA1S Is Altered by
Introducing or Eliminating Negative Charge at Residues 17 and
65--
Basic and large hydrophobic amino acids in prototypic PSDs
cooperatively promote association of proteins with plasma membrane through a combination of electrostatic and apolar interactions (43,
44). A high density of positive charge provided by clustered Arg and
Lys residues (e.g. see residues 11-25, 61-77, Fig.
6A) enables binding with anionic head groups of
phospholipids. The hydrocarbon interior of the membrane bilayer
simultaneously accommodates and stabilizes insertion of side chains of
large hydrophobic amino acids that are critical components of PSDs (43,
44).
Mutated CKA1 transgenes were generated and then expressed in AV-12
cells to determine consequences of introducing or eliminating negative
charge in PSD1 and PSD2 (Fig. 6A). A CKA1 protein that contains Ser17 to Ala and Ser65 to Ala
substitutions is efficiently routed to the cell periphery, leaving only
a modest amount of the adapter in other cell compartments (Fig.
7A). In contrast, conversion
of both Ser17 and Ser65 to Glu residues (which
mimic phospho-Ser by carrying negative charge) promotes accumulation of
CKA1/CKA1S in the cytoplasm (Fig. 7C). The strikingly
distinct patterns of localization of
CKA1Ala17-Ala65 and
CKA1Glu17-Glu65 strongly suggest
(a) nonphosphorylated PSD regions of CKA1 govern targeting
of the adapter protein (and tethered PKC3) to plasma/membrane-cortical cytoskeleton and (b) N-terminal phosphorylation of
Ser17 and/or Ser65 disrupts crucial
electrostatic and hydrophobic bonds, elicits disengagement of CKA1 (and
presumably CKA1·PKC3 complexes) from a cell surface microenvironment,
and promotes re-distribution of the adapter to the cytoplasm and/or
cytoplasmic organelles. A key implication of these results is that the
location of the wild type adapter protein may be dynamically regulated
by N-terminal phosphorylation and dephosphorylation. Consistent with
this idea, the intracellular pattern of CKA1/CKA1S localization appears
to be an intermediate, equilibrium distribution (Fig. 7B)
between the two extremes represented by
CKA1Ala17-Ala65 and
CKA1Glu17-Glu65 (Fig. 7, A and
C). The wild type adapter is enriched at the cell periphery,
but CKA1/CKA1S is also evident in cytoplasm. Thus, physiological
signals that stimulate either phosphorylation or dephosphorylation of
CKA1 may control the (a) accessibility of PKC3 to target
substrate-effector proteins at the cell surface or in the cytoplasm and
(b) incorporation or release of PKC3 from multicomponent
signaling complexes in different cell compartments.
Association of CKA1/CKA1S with The Cell Periphery Is Enhanced by a
PKC Inhibitor and Disrupted by Phorbol Ester or Protein Ser/Thr
Phosphatase Inhibitors--
Mechanisms that govern phosphorylation of
Ser17 and Ser65 in situ remain to be
determined. PKC3 phosphorylates these residues in vitro and
(apparently) in transfected cells (that contain an elevated level of
the aPKC) (Fig. 6B). However, wild type CKA1/CKA1S is
enriched at the cell periphery in stably transfected cells (Fig.
7B). Moreover, both CKA1 and PKC3 are targeted to the
periphery of cells that were cotransfected with a molar ratio of
transgenes (CKA1:PKC3 = 3) that yields an excess of aPKC tethering
sites (45). These results suggest that phosphorylation of adapter protein by tethered endogenous aPKCs or modest amounts of
sequestered recombinant PKC3 are tightly regulated. This could be
achieved through the dominant (regulated) activity of protein
phosphatases, the presence of modulators that inhibit phosphorylation
of Ser17 and Ser65 via interactions with the
kinase or CKA1, or incorporation of adapter·aPKC dimers into a
multiprotein complex that sterically precludes interactions between the
PKC3 catalytic domain and the N terminus of CKA1. In addition, PSD1 and
PSD2 are exceptionally good substrates for Ca2+ and/or
DAG-activated PKCs.2 Thus,
another possibility is that Ser17 and/or Ser65
are in vivo targets for classical PKC or novel PKC
isoforms rather than aPKCs.
Effects of DAG-activated PKCs and protein Ser/Thr phosphatases on
intracellular targeting of CKA1 and CKA1S were assessed in AV-12 cells
that stably express the wild type adapters (Fig. 7B).
Incubation of cells with TPA (12-tetradecanoyl phorbol 13-acetate), a
phorbol ester that selectively activates endogenous
DAG-dependent PKCs, caused substantial depletion of adapter
protein from the cell surface and promoted a concomitant increase in
the cytoplasmic content of CKA1/CKA1S (Fig.
8A). Treatment with a pair of
protein Ser/Thr phosphatase inhibitors (okadaic acid and cantharidin) or a combination of these inhibitors with TPA completely eliminated the
enriched pool of CKA1/CKA1S at the cell periphery and elicited a
homogeneous dispersal of adapter protein in cytoplasm (and/or internal membranes) (Fig. 8, B and C). Thus,
compounds that are expected to promote phosphorylation of
Ser17 and Ser65 elicit a pattern of CKA1/CKA1S
distribution that was previously observed for the CKA1
Glu17-Glu65 mutant (Fig.
7C). These observations support the idea that incorporation of phosphate at Ser17 and Ser65 in CKA1 (or the
equivalent of Ser65 in CKA1S) serves as a molecular switch
that enables dissociation of PKC3 adapters from the vicinity of plasma
membrane. In contrast, GF-109203X, an inhibitor of
DAG-dependent PKCs, enhanced accumulation of CKA1/CKA1S at
the cell surface and diminished the level of cytoplasmic adapter
protein (Fig. 8D). Inhibition of endogenous PKCs, which is
expected to reduce phospho-Ser17 and
phospho-Ser65 content in CKA1, yields an intracellular
pattern of adapter protein localization that was previously documented
for the CKA1 Ala17-Ala65 mutant (Fig.
7A). This result is consistent with the concept that
dephosphorylated PSDs play a central role in targeting/anchoring CKA1/CKA1S to plasma membrane. Concerted binding of Arg and Lys with
anionic plasma membrane phospholipids and insertion of co-clustered large hydrophobic side chains into the apolar interior of the lipid
bilayer are evidently optimized when negative charge is reduced or
excluded at residues 17 and 65.
The predicted relationship between intracellular location and status of
PKC phosphorylation sites in CKA1/CKA1S was directly assessed. In
control (untreated) AV-12 cells, adapter proteins accumulate at the
cell periphery. CKA1 and CKA1S isolated from untreated AV-12 cells are
excellent substrates for purified PKCs (Fig. 8E, lanes 1 and
2). Thus, PKC target sites are available in adapter proteins
associated with the cell periphery. When cells are incubated with
protein Ser/Thr phosphatase inhibitors, CKA1 and CKA1S are uniformly
distributed throughout the cytoplasm (Fig. 8B). Purified
PKCs catalyzed only minimal phosphorylation of immunoprecipitated adapter proteins derived from the cytoplasmic compartment (Fig. 8E, lanes 3 and 4). Thus, most of the PKC target
sites in cytoplasmic CKA1/CKA1S are phosphorylated in
situ.
Distribution of CKA1/CKA1S between the cell periphery and cytoplasm
appears to be dynamically regulated by levels and activities of
endogenous DAG-stimulated PKCs and opposing protein Ser/Thr phosphatases in a model cell system. Further development of this model
will entail the following: (a) identification of individual DAG-activated PKC isoforms that control adapter protein localization and (b) rigorous testing of the idea that phosphorylation of
Ser17 and/or Ser65 in CKA1 constitutes a key
regulatory switch in intact cells. Nevertheless, the currently
available data suggest a novel and potentially important consequence of
this mode of regulation. The localization and function of an atypical
PKC may be rapidly altered by hormones or growth factors that activate
phospholipases C
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
and Caenorhabditis
elegans PKC3 (1-3), are involved in transmitting mitogenic,
inflammatory, and anti-apoptotic signals; regulating gene expression;
controlling vesicular trafficking and ion channel activities;
establishing cell polarity; and mediating asymmetric cell divisions
(4-12). To exert control over such diverse aspects of cell
physiology, aPKCs regulate effector proteins in cytoplasm,
cytoskeleton, nucleus, and at the surfaces of plasma and internal
membranes (4-14). aPKCs lack structural features that mediate direct
association with cytoskeleton or organelles. Thus, elucidation of
alternative mechanisms that enable aPKCs to encounter and control
effector proteins in discrete microenvironments is an important goal.
Recent investigations (reviewed in our companion paper (45)) suggest
that aPKC functions are diversified and specialized via interactions
with adapter proteins (6, 14-20). Candidate adapter proteins possess
two fundamental features: a tethering domain that ligates an aPKC, and
a distinct targeting region that routes the adapter·aPKC complex to
intracellular sites enriched in substrate-effector proteins and/or
regulatory molecules that modulate phosphotransferase activity. The
aPKC "recruitment" model further suggests that systematic
characterization of aPKC adapter proteins will ultimately yield novel
insights into regulatory properties, substrate specificities, and
precise physiological roles of atypical PKCs.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]ATP (100-200 cpm/pmol), 5 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, 20 µg/ml phosphatidylserine). After a
4-6-min incubation at 30 °C, reactions were terminated by adding
0.2 volume of 5× gel loading buffer and proteins were
size-fractionated by denaturing electrophoresis (10% gel) (36).
Phosphorylated PSD1' and PSD2' proteins were visualized by
autoradiography. Radiolabeled proteins were excised from the gel, and
32P radioactivity was measured in a scintillation counter.
Km and Vmax values for PSD1'
and PSD2' were calculated from measurements of the rate of
incorporation of 32P radioactivity into the fusion proteins
(see legend for Fig. 6). Mutant PSD1' and PSD2' proteins were generated
by site-directed mutagenesis, expressed in E. coli,
purified, and phosphorylated as described above.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
6,
7,
9,
10,
12, and
13 and Fig. 1B, 111-233, 175-233,
and 212-233). Only mutations that deleted all or part of a
segment of PKC3 bounded by Gly212 and Ile224
abolished tethering with the PTB module (Fig. 1A,
8-
11 and Fig. 1B,
compare 212-233, 111-227, and 212-224 with
175-210 and 111-218). Thus, amino acids within
a contiguous 13-residue segment of PKC3 (Fig. 1D) (3))
apparently promote formation of adapter protein·PKC3 complexes.
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Fig. 1.
Mapping a segment of PKC3 that binds with the
PTB domain of CKA1/CKA1S. A, the indicated fragments of
PKC3 polypeptide were expressed as GAL4 DNA binding domain fusion
proteins and were tested for their abilities to interact with the CKA1
PTB domain via yeast two-hybrid complementation assays (++ indicates
that a strong signal was produced by transformed yeast within 30 min of
initiation of the -galactosidase assay). The N- and C-terminal
boundaries of the fragments are marked with numbers that correspond to
positions of amino acids within the full-length sequence of PKC3 (3)
(GenBankTM accession number AF02566). B shows
typical data obtained when yeast was cotransformed with a wild type
CKA1 gene (target vector) and either wild type or truncated
PKC3 transgenes (bait vector). The numbers indicate the N-
and C-terminal boundaries of PKC3 polypeptides encoded by the
recombinant bait vectors. Yeast colonies were grown on nitrocellulose
filters and assayed for protein interaction-dependent
expression of
-galactosidase. Cleavage of the
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal)
substrate generates an insoluble precipitate. Thus, positive colonies
appear as irregular black spots in photographs. All colonies that
expressed either wild type PKC3 or fragments of PKC3 that include amino
acids 111-233, 111-227, 175-233, 211-233, or 212-224 exhibited
-galactosidase activity; none of the colonies that contained PKC3
fragments encompassing amino acids 175-210 or 111-218 expressed
-galactosidase. C presents a diagrammatic representation
of the domain organization of PKC3. V1-V3 are
variable regions that are not conserved in other PKC isoforms.
D shows the amino acid sequence of the segment of PKC3 that
couples with CKA1/CKA1S (PTB domain).
promoter
and a GST gene. This enables expression of GST fusion proteins in
transfected cells. Complementary DNAs encoding amino acids 111-218,
111-227, or 111-233 from PKC3 were cloned into the pEBG vector. Each
recombinant pEBG construct and a CKA1S transgene were then introduced
into hamster AV-12 cells. Transfected cells accumulated similar amounts
of each GST partial PKC3 fusion protein (Fig.
2A, lanes 2-4) and CKA1S (not
shown). PKC3 fusion proteins were isolated and purified from cell
extracts by binding with GSH-Sepharose 4B beads. The CKA1S adapter
polypeptide (apparent Mr = 64,000) (45) was
coisolated with partial PKC3 proteins that contain residues 212-227
(Fig. 2B, lanes 3 and 4). However, GST-PKC3,
which lacks amino acids 219-224, and GST alone failed to complex CKA1S
(Fig. 2B, lanes 1 and 2).
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Fig. 2.
The candidate CKA1/CKA1S binding region of
PKC3 ligates an adapter protein in intact cells. AV-12 cells were
cotransfected with transgenes encoding CKA1S and either a GST partial
PKC3 fusion protein or GST alone. Cell proteins were extracted with
buffer containing 0.5% Triton X-100 24 h after transfection.
A, samples (40 µg) of extracted soluble proteins were
size-fractionated by denaturing electrophoresis, and resolved
polypeptides were transferred to an Immobilon P membrane. The blot was
probed with affinity purified IgGs directed against GST (1:2000
relative to serum). After incubation with secondary antibodies coupled
to peroxidase, antigen·IgG complexes were detected by an enhanced
chemiluminescence procedure (34). Signals recorded on x-ray film are
shown. Lane 1 received proteins derived from cells
transfected with the GST transgene. Lanes 2-4 contained
proteins isolated from cells transfected with GST-PKC3-(111-233),
GST-PKC3-(111-218), and GST-PKC3-(111-227) transgenes, respectively.
These recombinant genes encode chimeric proteins in which the C
terminus of GST is extended by fusion with segments of the PKC3
polypeptide that include amino acids 111-233, 111-218, or 111-227
(3). Both the fusion protein and GST alone are evident in lane
2. Only the relevant portion of the blot is shown. No other
immunoreactive bands were observed. Similar amounts of CKA1S were
detected in each batch of cotransfected cells (data not shown).
B, samples (300 µg) of proteins extracted from
cotransfected cells described in A were incubated with
GSH-Sepharose 4B beads for 2 h at 4 °C to isolate GST-PKC3
fusion polypeptides and tightly bound partner proteins. After extensive
washing, sequestered proteins were eluted from the beads and analyzed
by Western immunoblotting as described previously (3, 34). The blot was
probed with affinity-purified IgGs directed against CKA1/CKA1S (1:4000,
relative to serum). CKA1S polypeptide that coprecipitated with PKC3
fusion proteins was visualized via peroxidase-coupled secondary
antibodies and an enhanced chemiluminescence procedure. A detailed
description of the preparation, properties, and specificities of the
antibodies is provided in an accompanying paper (45). No signals were
obtained when blots were probed with either preimmune IgGs or anti-CKA1
IgGs in the presence of excess purified antigen. The ligands used for
binding with CKA1S were GST fusion proteins that contained residues
111-218 (lane 2), 111-227 (lane 3), and
111-233 (lane 4) from PKC3. Unmodified GST (lane
1) was used as a control.
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Fig. 3.
A synthetic peptide ligand inhibits tethering
of PKC3 by CKA1 and CKA1S. AV-12 cells were transfected with
transgenes encoding either CKA1/CKA1S or a GST fusion protein that
contains residues 111-227 from PKC3 (GST-PKC3-(111-227)). Samples (50 µl, 60 µg of protein) of extracted soluble proteins from cells
expressing CKA1 and CKA1S were mixed with an equal volume of extract
(75 µg of protein) derived from cells synthesizing
GST-PKC3-(111-227). The mixtures were incubated for 2 h at
4 °C in the presence of the indicated concentrations of a 15-mer
peptide (Genemed Inc.) whose sequence (SGGGIDNGAFHEHEI, designated
CBSP) corresponds to amino acids 210-224 in PKC3 (3). Incubations were
also performed with a randomly scrambled 15-mer peptide (Genemed Inc.)
that has the same amino acids arranged in a distinct sequence
(AHNIFGISGEHGEDG). Partial PKC3·adapter protein complexes were
isolated on GSH-Sepharose 4B beads and analyzed by Western immunoblot
analysis as described under "Experimental Procedures" and the
legend for Fig. 2. The blot was probed sequentially with
affinity-purified IgGs directed against CKA1/CKA1S (45) and secondary
antibodies coupled to peroxidase. IgG·CKA1/CKA1S complexes were
visualized by an enhanced chemiluminescence procedure. Only the
relevant portion of the blot is shown. No other immunoreactive bands
were observed. A shows competition by 50-2000
µM CBSP; B presents results from competition
assays that employed 0.5-50 µM CBSP.
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Fig. 4.
Identification of individual amino acids in
PKC3 that are essential for avid binding with the PTB domain of
CKA1/CKA1S. A, amino acids in the CKA1 binding region
of PKC3 were altered by site-directed mutagenesis as indicated under
"Experimental Procedures." Wild type and mutant GST fusions that
contain residues 111-227 from PKC3 were coexpressed with CKA1 and
CKA1S in transfected AV-12 cells. Binding of adapter proteins to mutant
and wild type GST partial PKC3 proteins was assessed by a combination
of affinity purification of the complexes on GSH-Sepharose 4B beads and
subsequent Western immunoblot analysis (see "Experimental
Procedures" and the legends for Figs. 2 and 3). Proteins eluted from
the beads were size-fractionated by denaturing electrophoresis,
transferred to an Immobilon P membrane, and probed with anti-CKA1/CKA1S
IgGs. Lane 1 received proteins coisolated with the wild type
CKA1 binding region of PKC3. Other samples include proteins coisolated
with CKA1 binding regions of PKC3 that were mutated as follows:
Ile214 to Ala (lane 2); Asn216 to
Ala (lane 3); Phe219 to Ala (lane 4)
and His222 to Ala (lane 5). IgG·CKA1 and
IgG·CKA1S complexes were visualized by enhanced chemiluminescence
methodology. Signals recorded on x-ray film are shown. B, a
Western blot probed with anti-GST IgGs reveals that similar amounts of
wild type and mutant GST-PKC3 fusion proteins were expressed and
isolated for each binding assay. Samples applied to lanes
1-5 are duplicates of samples used for lanes 1-5 in
A. Only the relevant portion of the blot is shown.
C, AV-12 cells were transfected with transgenes encoding
both CKA1 and PKC3. In addition, cells were cotransfected with a
recombinant pEBG vector that contained a transgene encoding either GST
partial PKC3 (amino acids 111-227) wild type protein (panel
1); GST partial PKC3 (amino acids 111-227) mutant protein
(Asn216 to Ala, panel 2); or GST alone
(panel 3). Western blots (probed with anti-GST IgGs)
revealed that transfected cells accumulate similar amounts of GST
fusion proteins or GST (data not shown). Cells were fixed,
permeabilized, and examined by confocal immunofluorescence microscopy
as previously reported (35). Fluorescence signals indicating the
intracellular location of full-length PKC3 were obtained by using
affinity-purified anti-PKC3 IgGs (3) and secondary antibodies tagged
with fluorescein isothiocyanate. Anti-PKC3 IgGs bind with epitopes
located between residues 477 and 597 at the C terminus of the atypical
kinase (3). These epitopes are not present in the competing GST partial
PKC3 fusion proteins. No signals were observed after blocking with
excess purified antigen or with preimmune serum.
- or
-helical turn conformation (27, 28), in which
side chains and main chains are oriented to complement a binding pocket
(surface) presented by the Numb-like,
-sandwich PTB domain of
CKA1/CKA1S (25-28, 45). Properties of the CKA1 PTB domain are
described in the accompanying paper (45). The PTB-binding segment of
PKC3 is embedded within the V2 "linker" region of the
kinase (Fig. 1C). The linker connects N-terminal pseudosubstrate and Cys-rich regulatory regions with the C-terminal phosphotransferase domain, thereby enabling physiological control of
enzyme activity (1-3). The discovery that the linker region contains a
site involved in tethering and targeting PKC3 to a specific
intracellular location reveals a second, previously unappreciated function for this segment of the atypical kinase.
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Fig. 5.
Comparison of peptide sequences that engage
the PTB domains of various signaling/regulatory proteins. Peptide
sequences of PTB domain ligands are aligned as described by Meyer
et al. (39). Residues thought to be essential for stable
binding of partner proteins with the indicated PTB domains are enclosed
in rectangles. The PTB domain ligands for Shc and IRS-1 PTB
domains contain phospho-Tyr, whereas other ligands are not
phosphorylated. In the consensus sequence X indicates any
amino acid; corresponds to an aromatic or large aliphatic
hydrophobic amino acid. Mutation of any of the residues marked with
asterisks abrogates the ability of PKC3 to bind with
CKA1.
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Fig. 6.
PKC3 phosphorylates Ser17 and
Ser65 in the PSDs of CKA1. A presents the
amino acid sequence of the basic N-terminal region of CKA1 (residues
1-89) (45). The sequence corresponding to PSD1 is marked with
asterisks; the sequence for PSD2 is underlined.
B, AV-12 cells were transfected with either transgenes encoding
both CKA1S and PKC3, the CKA1S transgene alone, or control expression
vector that lacked an insert. Twenty four hours after transfection,
proteins were extracted with buffer containing 0.5% Triton X-100.
Aliquots of extracted proteins (300 µg) were mixed with 2 µl of
affinity-purified IgGs directed against CKA1/CKA1S (final dilution of
1:50 relative to serum; preparation, properties, and specificity of the
antibodies are given in Ref. 3). After incubation for 3 h at
4 °C, antigen-antibody complexes were bound to protein A-Sepharose
4B beads and extensively washed by repeated dilution and centrifugation
(see "Experimental Procedures"). Finally, the beads were
resuspended in kinase reaction buffer that contained 15 ng of partially
purified PKC3 (3) and 50 µM [ -32P]ATP.
Samples were incubated for 15 min at 30 °C. Reactions were
terminated by dilution with 0.2 volume of 5× gel loading buffer and
heating at 95 °C for 5 min. Proteins were size-fractionated by
electrophoresis in a denaturing 10% polyacrylamide gel, and
32P-labeled polypeptides were visualized via
autoradiography on x-ray film. Lanes 1 and 2 received duplicate samples derived from cells expressing both CKA1S and
PKC3; lanes 3 and 4 contained duplicate samples
isolated from cells expressing only CKA1S; samples in lanes
5 and 6 were from cells transfected with control
expression vector (no cDNA insert). C, wild type
(WT) and mutant GST fusion proteins that include residues
1-63 (PSD1') or residues 59-108 (PSD2') from CKA1 were synthesized in
E. coli and purified to near-homogeneity by affinity
chromatography (see "Experimental Procedures"). Samples (2.5 µg)
of WT and mutant (Ser17 to Ala, Ser65 to Ala,
and Ser76 to Ala) PSD1' and PSD2' proteins were incubated
with 10 ng of purified PKC3 (3) and [
-32P]ATP for 20 min at 30 °C. Incorporation of 32P radioactivity into
the PSDs was monitored by denaturing electrophoresis, autoradiography,
and scintillation counting. Autoradiograms are shown. Typically, ~0.6
mol of phosphate was incorporated per mol of WT PSD1' or PSD2'.
D, kinetic analysis of PSD1' phosphorylation. Various
amounts of wild type GST-PSD1' protein were incubated with 10 ng of
purified PKC3 and [
-32P]ATP. After 6 min (linear
range) reactions were terminated, and the amount of 32P
radioactivity incorporated into PSD1' fusion protein was determined as
described under "Experimental Procedures." The data were best fit
(via computer algorithm) with the standard Michaelis-Menten equation.
Km and Vmax values derived
from the equation are shown. E, kinetics of PSD2'
phosphorylation. Assays and analysis were performed as described in
D above, except the reaction time was 4 min.
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Fig. 7.
Accumulation of CKA1/CKA1S at the cell
periphery is regulated by the introduction or elimination of negative
charge at residues 17 and/or 65. AV-12 cells were transfected with
a wild type CKA1 transgene (B) or transgenes encoding mutant
adapter proteins in which Ser17 and Ser65 were
substituted with Ala17 and Ala65 (A)
or Glu17 and Glu65 (C). Fixation and
permeabilization of cells and confocal immunofluorescence microscopy
were performed as previously reported (35). Fluorescence signals
indicating the intracellular locations of wild type and mutant CKA1
proteins were obtained by using anti-CKA1 IgGs and secondary antibodies
tagged with fluorescein isothiocyanate. No signals were observed after
blocking with excess purified antigen or with preimmune serum.
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Fig. 8.
Accumulation of CKA1/CKA1S at the cell
periphery is enhanced by a PKC inhibitor and disrupted by phorbol
ester. AV-12 cells that stably express a wild type CKA1 transgene
were incubated with 1 µM TPA (A); 500 nM okadaic acid plus 10 µM cantharidin
(B); a combination of 1 µM TPA, 500 nM okadaic acid, and 10 µM cantharidin
(C); or 5 µM GF109203X
(2-[(3-dimethylaminopropyl)-indol-3-yl]-3-(1H)-indol-3-yl)
maleimide), a potent PKC inhibitor (D). The intracellular
distribution of CKA1/CKA1S was then determined by confocal
immunofluorescence microscopy as described by Li et al.
(35). E, AV-12 cells that stably express CKA1 and CKA1S were
incubated with 10 µM cantharidin plus 0.5 µM okadaic acid (for 20 min) or without drugs. Cells were
lysed in 20 mM Tris-HCl buffer, pH 7.4, containing 50 mM NaF, 5 µM cantharidin, and 20 nM microcystin. After centrifugation at 125,000 × g for 60 min at 4 °C, supernatant solutions (which
contain proteins from cytoplasm) were collected. Membrane-associated
CKA1/CKA1S proteins were solubilized from the pelleted particulate
fraction of AV-12 cells by extraction with lysis buffer supplemented
with 2% Triton X-100. Detergent-soluble proteins were collected in the
supernatant solution after a second round of centrifugation
(125,000 × g). CKA1 and CKA1S were immunoprecipitated
from samples (300 µg of total protein) of cytoplasmic or
detergent-soluble membrane proteins and then incubated with
[ -32P]ATP and purified PKC3 as described in the legend
for Fig. 6B. (Substitution of PKC3 with a mixture of highly
purified cPKCs (
,
and
, Calbiochem) yielded similar results.)
32P-Labeled polypeptides were size-fractionated by
denaturing electrophoresis and visualized on x-ray film (see Fig.
6B). Lanes 1 and 2 received duplicate
samples of adapter proteins derived from the particulate (membranes and
cytoskeleton) fraction of control cells; lanes 3 and
4 contained duplicate samples of cytoplasmic adapter
proteins derived from cells treated with cantharidin and okadaic acid.
Parallel Western immunoblot analyses showed that each lane of the gel
was loaded with similar amounts of CKA1/CKA1S proteins (data not
shown). Results presented in lanes 3 and 4 were
replicated when cytoplasmic CKA1/CKA1S proteins (~10% of total
CKA1/CKA1S) from control cells were analyzed. Membranes and
cytoskeleton from drug-treated cells contained negligible amounts of
adapter proteins.
or C
, thereby initiating DAG-mediated signal
transduction. In such a pathway DAG-activated PKCs would serve as
upstream regulators that channel signals carried by a lipid second
messenger to a DAG-independent kinase. A pertinent speculation is that
colocalization of sites of DAG synthesis and CKA1/CKA1S protein in
plasma membrane could promote targeted accumulation of DAG-activated
PKCs. When kinase activity exceeds local protein phosphatase activity,
N-terminal phosphorylation of CKA1 would proceed, thereby triggering
translocation of aPKC·adapter complexes. These possibilities are
topics of ongoing studies.
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ACKNOWLEDGEMENT |
---|
We thank Ann Marie Malone for expert secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM22792 (to C. S. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Waksman Institute, Dept. of Molecular Biology and
Biochemistry, Rutgers University, Piscataway, NJ 08854.
§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Forch. 229, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2505; Fax: 718-430-8922; E-mail: rubin@aecom.yu.edu.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M008991200
2 L. Zhang, H. Feng, and C. S. Rubin, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: aPKC, atypical protein kinase C; PKC, protein kinase C; DAG, diacylglycerol; CKA1, protein kinase C adapter 1 (apparent Mr = 75,000); CKA1S, protein kinase C adapter 1 short isoform (apparent Mr = 64,000); PTB domain, phosphotyrosine binding domain; PSD, phosphorylation site domain; TPA, 12-tetradecanoyl phorbol 13-acetate; MARCKS, myristoylated alanine-rich C kinase substrate; GST, glutathione S-transferase.
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