Download Specific Interaction of the PDZ Domain Protein PICK1 with the

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 272, No. 51, Issue of December 19, pp. 32019 –32024, 1997
Printed in U.S.A.
Specific Interaction of the PDZ Domain Protein PICK1 with the
COOH Terminus of Protein Kinase C-a*
(Received for publication, July 30, 1997, and in revised form, September 18, 1997)
Jeff Staudinger‡§, Jianrong Lu¶, and Eric N. Olson¶i
From the ‡Department of Molecular Endocrinology, GlaxoWellcome, Research Triangle Park, North Carolina 27709 and
the ¶Department of Molecular Biology and Oncology, The University of Texas, Southwestern Medical Center at Dallas,
Hamon Center for Basic Cancer Research, Dallas, Texas 75235-9148
PICK1 is a protein kinase C (PKC) a-binding protein
initially identified using the yeast two-hybrid system.
Here we report that PICK1 contains a PDZ domain that
interacts specifically with a previously unidentified
PDZ-binding domain (QSAV) at the extreme COOH terminus of PKCa and that mutation of a putative carboxylate-binding loop within the PICK1 PDZ domain abolishes this interaction. The PDZ-binding domain in PKCa
is absent from other PKC isoforms that do not interact
with PICK1. We also demonstrate that PICK1 can homooligomerize through sequences that are distinct from
the carboxylate-binding loop, suggesting that self-association and PKCa binding are not mutually exclusive. A
Caenorhabditis elegans PICK1-like protein is also able
to bind to PKCa, suggesting a conservation of function
through evolution. Association of PKCa with PICK1 provides a potential mechanism for the selective targeting
of PKCa to unique subcellular sites.
Protein kinase C (PKC)1 a belongs to a family of serine/
threonine protein kinases that are activated in response to a
wide variety of hormones, mitogens, and neurotransmitters (1,
2). The PKC family is comprised of eleven isoforms, encoded by
10 genes. Individual cells typically express multiple isoforms of
PKC (3). Although purified PKC proteins can phosphorylate
many different substrates in vitro, individual PKC isoforms
appear to mediate distinct and separate functions in vivo, due
to their selective localization to different subcellular sites.
In unstimulated cells, PKCa resides primarily in the cytoplasm, and upon stimulation of cells with signals that promote
the hydrolysis of membrane phospholipids, it translocates to
the plasma membrane (4), as well as the nuclear envelope
(5–7). It has been proposed that the localization of PKC to
different intracellular sites is mediated by specific PKC-binding proteins that can discriminate between different PKC isoforms. A number of PKC binding proteins have been cloned and
characterized (8, 9), but the specific amino acid sequences
* This work was supported by grants from the Robert A. Welch
Foundation and the National Institutes of Health (to E. N. O.). The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
§ Performed portions of the work while a graduate student at the
University of Texas (Southwestern).
i To whom correspondence should be addressed: Dept. of Molecular
Biology & Oncology, University of Texas Southwestern Medical Center
at Dallas, 6000 Harry Hines Blvd., Dallas, TX 75235-9148. Tel.: 214648-1187; Fax: 214-648-1196; E-mail: [email protected].
1
The abbreviations used are: PKC, protein kinase C; PCR, polymerase chain reaction; AD, activation domain; PAGE, polyacrylamide gel
electrophoresis; DB, DNA-binding domain; GST, glutathione
S-transferase.
This paper is available on line at http://www.jbc.org
involved in interaction of these proteins with different PKC
isoforms have not been defined, nor has it been demonstrated
how various PKC-binding proteins might distinguish between
PKC isoforms.
Recent studies have identified the PDZ domain (PSD-95,
dishevelled and ZO1) as a modular protein-protein interaction
motif that serves to localize proteins to specific subcellular sites
(10 –14). This domain binds to proteins containing the tripeptide motif (S/T)XV (X 5 any amino acid) at their COOH termini
(10, 11, 15–22). PDZ domain-containing proteins have been
shown to mediate protein-protein interactions during receptor
and ion channel clustering and to recruit kinases and phosphatases to their membrane-associated substrates. Certain
PDZ domains have also been shown to homooligomerize,
thereby contributing to the formation of multiprotein complexes at specific subcellular sites (23, 24).
PICK1 is a PKCa-binding protein cloned using the yeast
two-hybrid system (25). Here we report that PICK1 contains a
PDZ domain at its NH2 terminus, which is required for interaction with a PDZ-binding motif (QSAV) at the extreme COOH
terminus of PKCa. This PDZ-binding motif is specific to PKCa
and can account for the selective interaction of PICK1 with
activated PKCa and not with other PKC isoforms. PICK1 can
also homooligomerize through its PDZ domain. We also describe a PICK1-like protein from Caenorhabditis elegans that
can function as a PKCa-binding protein, suggesting a conservation of function through evolution. We speculate that the
function of PICK1 is to localize activated PKCa to the plasma
membrane, thereby bringing the kinase in proximity with specific substrates and lending isoform specificity in vivo to the
kinase.
EXPERIMENTAL PROCEDURES
Expression Vectors—Construction of GAL4(DB)-PKC7 was described
previously (25, 26). The resulting plasmid fuses amino acids 302– 672 of
bovine PKCa to the DNA-binding domain (amino acids 1–147) of yeast
GAL4 in the yeast expression vector pAS1. GAL4(DB)-PKC7(D670 –
672) was constructed using PCR primers to remove the indicated amino
acids and introduce a BamHI site using GAL4(DB)-PKC7 as a template.
The amplified fragment contained an EcoRI restriction site from the
pAS1 vector at the 59 end and a termination codon preceding a BamHI
restriction site immediately following the glutamine at position 669 of
bovine PKCa. The fragment was digested with EcoRI and BamHI and
cloned back into pAS1 that had been digested with EcoRI and BamHI.
The GAL4(AD)-PICK1 K27A and GAL4(AD)-PICK1 KD27,28AA plasmids were constructed using the QuickChange site-directed mutagenesis kit (Stratagene) as directed. The GAL4(DB)-PKC7(QSAV669 –
672AAAA) mutation was generated by digesting pECE-FLAGPKC7(QSAV669 – 672AAAA) with BamHI and EcoRI and gel purifying
the 1.0-kilobase insert. This DNA fragment was cloned into pAS1 that
had been digested with EcoRI and BamHI. The GAL4(DB)-PICK1(1–
358) yeast expression construct was constructed using PCR primers to
introduce an NcoI site at amino acid 1 and a SmaI site following a
termination codon after amino acid number 358 in mouse PICK1 using
GAL4(AD)-PICK1 as a template. The resulting fragment was digested
with NcoI and SmaI and inserted into pAS1 that had been digested with
32019
32020
PICK1 PDZ Domain-PKCa Interaction
FIG. 1. Sequence comparison between mouse PICK1 and other PDZ domain proteins. A, the PDZ domain of PICK1 and other related
PDZ domain-containing proteins are shown. The putative carboxylate-binding loop, as described by Doyle et al. (29), is also indicated. Sources of
sequences were hROS1 (42), mPSD95 (43), dCAN (27), dDSH (44), hNOS (45), and hZO1 (46). B, comparison of the amino acid sequences of mouse
PICK1 and C. elegans PICK1-like protein. The PDZ domain is indicated in black.
NcoI and SmaI.
The GAL4(AD)-PICK1 deletions were constructed using PCR primers that introduced BglII restriction sites to amplify the respective
fragments using GAL4(AD)-PICK1 as a template. The resulting fragments were cloned into the BglII restriction site of pACT.
The C. elegans expressed sequence tag accession number Z14656 was
obtained from R. Waterston (Washington University), and the fulllength sequence was determined by automated sequencing. The C.
elegans PICK1 activation domain fusion expression vector was constructed by using lGT10 insert-screening amplimers (CLONTECH) to
amplify the fragment from the phage clone. The resulting fragment was
cloned into the pCRII plasmid (Invitrogen-TA cloning kit C2020 – 03)
and sequenced in its entirety. Using PCR primers that introduce BglII
restriction sites at the NH2 terminus and the COOH terminus, the
cDNA was amplified and cloned into pACT for expression in yeast. The
resulting plasmid fuses the full-length C. elegans PICK1 protein
COOH-terminal of the GAL4 activation domain (AD) in pACT for expression in yeast.
Two-hybrid Assays—Yeast two-hybrid assays were performed as described previously (25, 26). Interactions between bait and prey were
monitored by b-galactosidase activity in colonies on plates.
GST Binding Assays—Binding of in vitro translated PICK1 and PKC
to GST-PICK1 fusion protein was performed as described previously
(25). Briefly, 15 mg of GST-PICK1 bound to glutathione agarose beads
was incubated for 30 min at 4 °C with 20,000 cpm of 35S-labeled in vitro
translation products in 300 ml of incubation buffer (50 mM KCl, 40 mM
Hepes, pH 7.5, 5 mM mercaptoethanol, 0.1% Nonidet P-40). Beads were
then washed five times in 1 ml of NETN (250 mM NaCl, 20 mM Tris-Cl,
pH 8.0, 0.5 mM EDTA, 1% Nonidet P-40). After washing and removing
the final supernatant, the pellet was resuspended in SDS sample buffer
and resolved by SDS-PAGE.
Immunoprecipitations—To test for interactions between PICK1 and
PKC in vivo, COS cells were transiently transfected with 2 mg of
expression vectors encoding PICK1 and wild-type PKCa or PKC mutant
QSAV669 – 672AAAA. Two days later, cells were harvested and lysed on
ice in 0.5 ml of lysis buffer (50 mM Tris-Cl, pH 8.0, 50 mM NaCl, 1%
Nonidet P-40 with complete protease inhibitors (Boehringer Mannhein
1697– 498)). Following centrifugation for 10 min at 12,000 rpm in a
microfuge, the supernatant was removed, and 2 mg of nonimmune
mouse IgG was added, followed by incubation for 30 min at 4 °C. 30 ml
of protein A/G was then added for an additional 30 min, after which the
extract was centrifuged. The supernatant was removed, and 2 ml of
MC5 anti-PKCa monoclonal antibody (Amersham RPN.536) was added
and incubated for 30 min at 4 °C. 50 ml of protein A/G was then added,
and the extract was mixed for 30 min followed by centrifugation. The
resulting supernatant was washed twice with ice- cold lysis buffer and
centrifuged, and the supernatant was removed. The pellet was then
resuspended in 2 3 SDS sample buffer with dithiothreitol, boiled for 5
min, and separated by 10% SDS-PAGE. Proteins were then transferred
to nitrocellulose by Western blotting, and PICK1 was detected using a
rabbit polyclonal antibody raised against a GST-PICK1 fusion protein.
Detection was accomplished using goat anti-rabbit horseradish peroxidase-conjugated antibody and a chemiluminscence detection kit
(Amersham RPN.2108).
RESULTS
PICK1 Is a PDZ Domain-containing Protein—In searching
the data base for proteins related to PICK1, we discovered that
PICK1 contained a PDZ domain near its NH2 terminus (Fig.
1A). The PDZ domain of PICK1 is most closely related to those
of the receptor tyrosine kinase ROS1, the third PDZ domain in
the post-synaptic density protein PSD95, and the Drosophila
protein canoe, which has been implicated in wingless signaling
(27). The PICK1 PDZ domain is also similar to the C. elegans
PICK1 PDZ Domain-PKCa Interaction
32021
FIG. 2. Specificity of PICK1 for interaction with PKCa in the yeast two-hybrid system. A, the COOH-terminal regions in PKCa (also
referred to as PKC7 in B and C), PKCbI, and PKCe were fused to the GAL4 DNA-binding domain and tested for interaction with PICK1 fused to
the GAL4 activation domain using the yeast two-hybrid system. The COOH terminus of PKCa interacted strongly with PICK1, whereas no
interaction was detected for the other PKC isoforms. B, the COOH-terminal residues of PKCa (QSAV) in PKC7 were deleted or mutated to
alanines, and the resulting mutants were tested for their abilities to interact with PICK1 in the yeast two-hybrid system. Neither of the mutants
were able to interact with PICK1. C, the complete C. elegans PICK1-like protein was fused to the GAL4 activation domain and tested for interaction
with the wild-type and mutant PKCa proteins fused to the GAL4 DNA-binding domain. The locations of the variable (V) and constant (C) regions
of the PKC isoforms are indicated.
LIN-7 PDZ domain, which has been shown to mediate clustering of the receptor tyrosine kinase LET-23 (28). The PDZ domain in PICK1 appears to be relatively divergent when compared with other PDZ domains in general.
As we reported previously (25), the sequence of mouse PICK1
shares significant homology with an expressed sequence tag
from C. elegans. We therefore obtained this cDNA and sequenced it in its entirety. Mouse PICK1 protein shares 44%
identity and 63% similarity with the corresponding C. elegans
protein (Fig. 1B). There are two primary regions of high identity between the two proteins. The first region includes the PDZ
domain, which is 47% identical, as well as adjacent sequences.
The carboxylate-binding loops of the two proteins, which in
other PDZ domain proteins have been shown to mediate binding to the COOH-terminal carboxyl group of PDZ-binding proteins, are similar but not identical in the C. elegans and mouse
proteins. The other region of high identity begins at amino acid
244 and ends at amino acid 331 in mouse PICK1. Mouse PICK1
protein contains a stretch of nine acidic amino acids (residues
382–390) at the COOH-terminal end of the protein that are not
highly conserved in the C. elegans protein, although the homologous region is acidic overall.
Detection of PKC-PICK1 Interactions Using the Yeast Twohybrid System—Our previous studies showed that PICK1 interacted with the catalytic region of PKCa (25). To determine
whether PICK1 could also interact with other PKC isoforms,
we used the hinge and catalytic regions of PKC bI and PKCe
fused to the GAL4 DNA-binding domain (DB) as bait in the
yeast two-hybrid system and tested for interaction with
GAL4(AD)-PICK1, in which the GAL4 activation domain was
fused to mouse PICK1. Potential interaction was tested by
expressing the plasmids in yeast and assaying for GAL4-dependent reporter activity (Fig. 2A).
All of the GAL4 fusion proteins were expressed at comparable levels as determined by Western blots of yeast extracts
transformed with the respective expression plasmids, using a
monoclonal antibody against the GAL4 DNA-binding domain
(data not shown). None of the GAL4 fusion proteins activated
expression of the reporter gene alone (data not shown).
GAL4(AD)-PICK1 strongly activated expression in the presence of the PKCa catalytic domain, referred to as PKC7,
whereas it failed to activate reporter gene transcription in the
presence of PKC bI- or PKCe-GAL4 DNA-binding domain fusion proteins (Fig. 2A). These results suggested that PICK1
was an a isoform-specific binding protein.
Characterization of the PKCa PICK1-binding Domain—Because PDZ proteins bind the consensus sequence ((S/T)XV) at
the extreme COOH terminus of proteins, we examined PKCa
for this sequence. Indeed, the COOH-terminal sequence of
PKCa from every species in which it has been cloned is QSAV.
To determine whether this was the PICK1-binding domain of
PKCa, we deleted this region by introducing a stop codon
32022
PICK1 PDZ Domain-PKCa Interaction
FIG. 3. Effects of carboxylate-binding loop mutations in PICK1 on binding to PKCa in the yeast two-hybrid system. Wild-type
PICK1 or PICK1 point mutants in the carboxylate-binding loop were fused to the GAL4 activation domain and tested for interaction with the
catalytic domain of PKCa (called PKC7) fused to the GAL4 DNA-binding domain using the yeast two-hybrid system. The acidic region near the
COOH terminus of PICK1 is indicated D/E.
following the glutamine at position 669 in PKCa. This mutant,
GAL4(DB)-PKC7(D670 – 672), failed to interact with PICK1 in
the yeast two-hybrid assay (Fig. 2B). We then mutated the four
COOH-terminal amino acids in PKCa to alanines. This mutant, GAL4(DB)-PKC7(QSAV669 – 672AAAA), also failed to interact with PICK1. These results indicate that the sequence
QSAV in PKCa, which corresponds to the consensus for PDZ
recognition, is required for interaction with PICK1.
To determine whether the C. elegans PICK1-like protein was
also a PKCa-binding protein, we tested whether it could interact with PKCa in the yeast two-hybrid system (Fig. 2C). Indeed, C. elegans GAL4(AD)-PICK1 was able to activate the
reporter gene only in the presence of GAL4(DB)-PKC7 and was
unable to interact with either of the mutants that lacked the
COOH-terminal PDZ-binding domain. These results show that
the C. elegans PICK1-like protein exhibits the same PKCbinding properties as mouse PICK1.
Requirement of the Putative Carboxylate-binding Loop of
PICK1 for Binding to PKCa—The carboxylate-binding loop is a
7– 8-amino acid segment near the NH2 terminus of the PDZ
domain that interacts directly with the COOH-terminal peptides of PDZ-binding proteins (17). The first residue of a carboxylate-binding loop (amino acid 27 in mouse PICK1) is almost always either an arginine or lysine (Fig. 1A) that interacts
with the carboxylate via a highly ordered water molecule (29).
Additional hydrogen bonds are established between the carboxylate oxygens and other residues in the binding loop.
To test the importance of the carboxylate-binding loop of
PICK1 for PKCa binding, we mutated lysine 27 alone or together with aspartic acid 28 to alanines (Fig. 3). The lysine 27
to alanine mutant (GAL4(AD)-PICK1K27A) was able to interact with GAL4(DB)-PKC7 in yeast, whereas the double mutant
(GAL4(AD)-PICK1KD27,28AA) was not. These results show
that the putative carboxylate-binding loop of the PICK1 PDZ
domain is involved in interaction with PKCa.
Detection of PICK1 Homooligomerization—Because several
PDZ domains have been reported to self-associate (24), we
examined whether PICK1 could homooligomerize through its
PDZ domain by fusing amino acids 1–358 of PICK1 to the
GAL4 DNA-binding domain and assaying reporter gene activity in the presence of specific deletions of PICK1 fused to the
GAL4 activation domain (Fig. 4).
Full-length GAL4(AD)-PICK1 showed a strong interaction
with GAL4(DB)PICK1(1–358). A COOH-terminal deletion mutant of PICK1 lacking the acidic domain (PICK1/1–358) also
interacted strongly with GAL4(DB)PICK1(1–358). Residues
1–59 or 231–358 were unable to interact with GAL4(DB)PICK1(1–358), whereas residues 13–358 showed a strong interaction.
These results indicated that PICK1-PICK1 interactions were
mediated by the region of the protein containing the PDZ
domain. We have been unable to obtain stable expression in
yeast of shorter regions of the protein containing only the PDZ
domain.
To determine whether the carboxylate-binding loop, which
mediates interaction of PICK1 with PKCa, was also required
for PICK1 homooligomerization, we tested the PICK1 mutants
in which residues 27 and 28 were changed to alanine. Both
mutants, GAL4(AD)-PICK1(K27A) and GAL4(AD)-PICK1(KD27,28AA), were able to homooligomerize with GAL4(DB)PICK1(1–358). The finding that mutations in the putative carboxylate-binding loop of PICK1 (Lys27-Asp28), which disrupted
interaction with PKCa, had no effect on homooligomerization
suggests that PICK1 may be able to homooligomerize at the
same time it is bound to PKCa.
In Vitro Binding PICK1 Assays—To determine whether the
PICK1 interactions observed with the yeast two-hybrid assays
could also occur in vitro, we tested the ability of a GST-PICK1
fusion protein to interact with [35S]methionine-labeled PKC7
and PICK1 obtained from in vitro translation. As shown in Fig.
5A, when GST-PICK1 was incubated with labeled PKC7 and
precipitated with glutathione agarose beads, the interaction
between the two proteins was readily detected. In contrast,
GST-PICK1 failed to interact with the PKC7 mutant
QSAV669 – 672AAAA, in which the COOH-terminal PDZ-binding motif was eliminated. GST alone did not interact with
PKC7 further demonstrating the specificity of the interaction.
GST-PICK1 also interacted with the 35S-labeled PICK1 in
vitro translation product, as well as with the PICK1 mutants
K27A and KD27,28AA, in the yeast two-hybrid assay (Fig. 5B).
GST alone did not interact with PICK1. Thus, the specificity of
the PICK1-PKCa and PICK1-PICK1 interactions observed
with the yeast two-hybrid system is maintained in vitro.
Detection of PICK1-PKCa Interactions in Transfected
Cells—We also tested whether binding of PICK1 to PKCa in
mammalian cells required the PDZ-binding motif of PKCa, as
was the case in the two-hybrid and GST-PICK1 binding assays.
For these experiments, COS cells were transfected with expression vectors encoding PICK1 and either wild-type PKCa or
PKCa mutant QSAV669 – 672AAAA using a PKCa-specific
monoclonal antibody under low salt conditions. Following separation of immunoprecipitation reactions by SDS-PAGE, proteins were transferred to nitrocellulose, and PICK1 protein was
detected by Western blot with a rabbit polyclonal antibody
against PICK1. PICK1 was detected in extracts of cells transfected with wild-type PKCa but not with the mutant nor in
mock transfected cells (Fig. 6A). The failure to detect PICK1 in
PICK1 PDZ Domain-PKCa Interaction
32023
FIG. 4. Detection of PICK1 homooligomerization in the yeast two-hybrid system. A series of PICK1 deletion and point mutants fused
to the GAL4 activation domain were tested for their abilities to interact with amino acids 1–358 of PICK1 fused to the GAL4 DNA-binding domain
using the yeast two-hybrid system.
FIG. 5. Detection of PICK1 interactions in vitro. A, PKC7 or PKC7 mutant QSAV669 – 672AAAA (PKC7 MUT) was translated in a rabbit
reticulocyte lysate in the presence of [35S]methionine. Labeled products were then incubated with GST or GST-PICK1 and precipitated with
glutathione agarose, as described under “Experimental Procedures.” Labeled proteins were resolved by SDS-PAGE. Labeled PKC7 and PKC7
mutant proteins were also loaded directly onto the gel in adjacent lanes for comparison. The amount of labeled proteins used for the GST binding
experiments in lanes 1–3 was four times more than the amount loaded in lanes 4 and 5. B, PICK1 or the indicated PICK1 mutants were translated
in a rabbit reticulocyte lysate in the presence of [35S]methinone and then incubated with GST or GST-PICK1 as described in A. The amount of
labeled proteins used in the GST binding experiments in lanes 1– 4 was four times more than the amount loaded onto lanes 5–7. Molecular mass
markers are indicated at the left.
extracts from cells transfected with the mutant form of PKCa
was not due to a failure of this mutant to accumulate because
Western blots of cells transfected with wild-type or mutant
PKC showed equivalent amounts of protein (Fig. 6B). We conclude that the PDZ-binding motif of PKCa is required for interaction with PICK1 in mammalian cells, just as it is in yeast.
DISCUSSION
Our results show that PICK1 contains a PDZ domain that is
required for interaction with the COOH terminus of PKCa.
PICK1 is also able to homooligomerize through its PDZ domain, but mutations in the putative carboxylate-binding loop
that abolish binding to PKCa have no effect on homooligomerization. The finding that determinants for interaction of PICK1
with itself and with PKCa are separable suggests that PICK1
can simultaneously self-associate and interact with PKCa. The
fact that the extreme COOH terminus of PKCa mediates binding to PICK1 also suggests that the kinase can be catalytically
active when bound to PICK1.
Among the vertebrate PKC isoforms, the consensus sequence
for PDZ binding is contained only in PKCa. Thus, the specific
interaction of this sequence with PICK1 provides a molecular
FIG. 6. Detection of PICK1 interactions in cell extracts. A, COS
cells were transfected with expression vectors encoding PICK1 and
wild-type PKCa (lane 3) or PKCa mutant QSAV669 – 672AAAA (lane 2).
Extracts were prepared from transfected cells and were immunoprecipitated with anti-FLAG antibody, which recognized the transfected
PKC proteins. Following separation of immunoprecipitates by SDSPAGE, Western blotting was performed using an anti-PICK1 antibody.
The PICK1 protein, migrating at about 50 kDa, can be observed only in
the lane containing wild-type PKCa and PICK1. Other bands are nonspecific background due to the relatively low affinity of the anti-PICK1
antibody. Lane 1 was performed with untransfected cells. B, to ensure
that wild-type and mutant PKC accumulated to equivalent levels in
transfected cells, Western blots were performed using anti-FLAG antibody. Molecular mass markers are indicated to the left.
PICK1 PDZ Domain-PKCa Interaction
32024
mechanism for the selective targeting of PKCa to distinct subcellular sites, thereby allowing specific phosphorylation events
to occur. In Drosophila, the subcellular location and substrate
specificity of eye-specific PKC(InaC) has been shown to be
regulated by the PDZ domain-containing protein InaD (30).
The COOH terminus of InaC contains the sequence ITII, which
is compatible with PDZ binding (17).
Our results show that the C. elegans PICK1-like protein
recognizes the COOH terminus of mammalian PKCa. There
have been several PKC proteins identified thus far in C. elegans, but none contain a PDZ-binding domain at their COOH
terminus. This suggests either that additional, as yet unidentified PKC proteins exist in this organism or that this PICK1like protein interacts with other PDZ-binding proteins.
It will be of interest to define the spectrum of proteins that
interact with PICK1. It is possible that PDZ-PDZ interactions
may enable PICK1 to interact with other PDZ domain-containing proteins. The NMDA receptor, for example, which clusters
specifically at the postsynaptic density in the neuronal synapse, binds to PSD-95 and has been reported to be regulated by
PKC activity (31, 32). The a-amino-3-hydroxy-5-methylisoxazole-4-propianic acid receptor, which also clusters at the post
synaptic density through interaction with a PDZ containing
protein (33), has also been reported to be regulated by PKC
activity (34, 35).
PICK1 appears to be one of many PKC-binding proteins
(36 – 41). Whether interaction of PKCa with each of these proteins is mutually exclusive and how these different binding
proteins influence the subcellular localization, substrate specificity, and protein-protein interactions of PKCa represent interesting issues for the future.
Acknowledgment—We thank A. Tizenor for assistance with graphics.
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