(Received for publication, December 29, 1995)
From the
Compartmentalization of the type II cyclic AMP-dependent kinase
(PKA) is achieved through association of the regulatory subunit (RII)
with A-kinase anchoring proteins (AKAPs). Using an interaction cloning
strategy with RII as a probe, we have isolated cDNAs encoding a
novel 1129-amino acid protein that contains both a PKA binding region
and a peroxisome targeting motif. Northern analysis detected mRNAs of
9.7 and 7.3 kb in several rat tissues with the highest levels present
in the brain and testis. Western analysis and RII overlay experiments
showed that the protein is approximately 220 kDa and was, therefore,
named AKAP 220. Immunoprecipitation of AKAP 220 from rat testis
extracts resulted in co-purification of the type II PKA holoenzyme. The
specific activity of PKA increased 458-fold from 7.2 pmol/min/mg in the
cell lysate to 3.3 nmol/min/mg in the immunoprecipitate.
Immunohistochemical analysis of rat testicular TM4 cells showed that
AKAP 220 and a proportion of RII were co-localized in microbodies that
appear to be a subset of peroxisomes. Collectively, these results
suggest that AKAP 220 may play a role in targeting type II PKA for
cAMP-responsive peroxisomal events.
Intracellular responses of many hormones are mediated by signal
transduction pathways that alter the phosphorylation state of key
regulatory proteins(1) . Protein phosphorylation is a
reversible process involving two classes of signaling enzymes: protein
kinases, which catalyze the phosphotransfer reaction, and
phosphoprotein phosphatases that catalyze
dephosphorylation(1, 2) . The activity of both enzyme
classes is tightly regulated and responds to fluctuations in diffusible
second messengers such as Ca, phospholipid, and
cAMP(1) . The predominant effect of cAMP is to activate a
cAMP-dependent protein kinase (PKA) (
)that modifies serine
and threonine residues on substrate proteins(3) . Since PKA has
a broad substrate specificity and is present in relatively high
intracellular concentrations(4) , it is critical to restrict
the activity of the enzyme and its substrate availability. One way to
accomplish this is by localizing PKA to specific cellular
compartments(5) . Compartmentalization of the kinase seems to
represent a regulatory mechanism that could increase the selectivity
and intensity of a cAMP-mediated hormonal response. This is achieved,
in part, through the association of the kinase with a family of
A-kinase anchoring proteins (AKAPs)(6) .
In recent years, numerous AKAPs have been identified that target PKA to the plasma membrane, cytoskeleton, endoplasmic reticulum, Golgi, mitochondria, and nuclear matrix(7, 8, 9, 10, 11, 12, 13, 14, 15) . These proteins represent a growing family of signaling molecules that contain a conserved PKA binding motif and function to localize the kinase to particular subcellular sites(16) . In this report, we describe the cloning and characterization of another AKAP, called AKAP 220. This protein associates with PKA in testis where it may target the kinase to peroxisomes.
Figure 1:
Sequence of AKAP 220. A, the
nucleotide sequence and the deduced amino acid sequence of the cDNA
encoding the A-kinase anchoring protein, AKAP 220. The boxed area indicates the putative RII binding region, while the
peroxisome-targeting sequence is underlined and in italics. B, helical wheel representation of AKAP 220 (residues
905-918) drawn as an -helix of 3.6 amino acids/turn. The shaded area indicates the hydrophilic residues, and the black area indicates the hydrophilic residues. C,
sequence homology between AKAP 220 (residues 905-918) and the RII
binding regions of two other AKAPs, AKAP 150 and Ht 31. The shaded
area indicates amino acid identity and conserved amino acids are
indicated (*).
Figure 2:
The tissue distribution of the AKAP 220
mRNA. A, 2 µg of poly(A) RNA from rat
tissues (rat MTN, Clontech) were probed with a
P-radiolabeled 936-bp fragment excised from the 3` end of
GH
-12 as described under ``Experimental
Procedures.'' B, the same filter was probed with
P-radiolabeled
-actin. Hybridizing mRNA species were
detected by autoradiography. The tissue source of each RNA is indicated
above each lane. Kilobase markers are indicated beside each
panel.
The AKAP 220 nucleotide and protein sequence
were compared with the Genbank(TM) data base, and there was no
overall homology to known protein sequences. However, the last three
amino acids of the protein, Cys-Arg-Leu, fulfill the criteria for a
COOH-terminal peroxisomal (microbody) targeting signal (Fig. 1A). In addition, residues 905-918 are
likely to represent an RII binding site as this region exhibits high
probability for forming an amphipathic -helix (Fig. 1B) and shows limited homology to the RII binding
regions of other AKAPs (Fig. 1C).
Figure 3:
Recombinant AKAP 220 fragment specifically
binds RII. A fragment of the AKAP 220 cDNA (encoding residues
761-1129 of the protein) was expressed using the pET16b bacterial
expression vector. Bacterial extracts, induced or uninduced (100
µg), or purified protein (10 µg) were separated by
electrophoresis on 10% (w/v) SDS-PAGE and electrotransferred to PVDF
membranes. A, gels were stained with Coomassie Blue dye. B, the recombinant AKAP 220 fragment was detected by Western
blot with affinity-purified antibodies. RII binding proteins were
detected by a solid-phase binding assay (22) using
P-radiolabeled RII
as a probe in the absence (C) or presence (D) of 1 µM anchoring
inhibitor peptide, Ht 31(493-515). Sample sources are indicated
above each lane, and the molecular weight markers are indicated beside
each panel.
Figure 4:
Estimation of the AKAP220 binding affinity
for RII. Binding of
P-radiolabeled RII
to the
COOH-terminal fragment of AKAP 220 (residues 761-1129) and a
corresponding fragment of the human thyroid anchoring protein Ht 31 was
measured by a semiquantitative overlay procedure. Aliquots of the
purified protein ranging from 5 to 125 ng were immobilized onto
nitrocellulose filters using a slot-blot apparatus. Individual filters
were probed with excess
P-radiolabeled RII
(specific
activity: 2.1-1.5
10
cpm/nmol). Detection of
immobilized RII
was by autoradiography. Quantitation of binding
over the range of protein concentrations was determined by densitometry
of the radiographs. Signals were normalized for the specific activities
of each RII
probe. Binding curves for AKAP 220 (
) and Ht 31
(
) are presented from three experiments; the standard deviation
of the integrated density is indicated.
Figure 5:
AKAP 220 is enriched in rat testis.
Detergent-solubilized extracts of several rat tissues (100 µg of
protein/lane) were separated on 7.5% SDS-PAGE and electrotransferred to
PVDF. A, the solid-phase overlay method was used to detect RII
binding proteins in these tissues. The blots were probed with P-radiolabeled RII
, and the RII-binding proteins were
detected by autoradiography. B, Western blot analysis was
performed on identical filters as described under ``Experimental
Procedures.'' The migration of an immunoreactive protein that
corresponds to AKAP220 is indicated by an arrow. Tissue
sources are indicated above each lane, and molecular weight markers are
indicated beside each panel.
Figure 6: AKAP 220 associates with type II PKA in rat testis. A, immunoprecipitation of the AKAP 220/PKA complex from rat testis was performed with 15 µg of affinity-purified AKAP 220 antibody as described under ``Experimental Methods.'' Fractions collected from the protein A-Sepharose column were separated by electrophoresis on a 7.5% SDS-PAGE gel and electrotransferred to PVDF. B, AKAP 220 was detected by Western blot. Sample sources are indicated above each lane. C, PKA activity was measured in all fractions by a filter paper assay using kemptide as a substrate. D, the catalytic subunit of PKA was specifically eluted from the immunoprecipitate with 0.5 mM anchoring inhibitor peptide, Ht 31(493-515), and was detected by Western blotting.
Figure 7: Immunocytochemical analysis of rat testicular cells (TM4). TM4 cells were fixed with picric acid/paraformaldehyde and incubated with anti-RII antibodies (A), anti-AKAP 220 antibodies (B), anti-peroxisome 70-kDa protein antibodies (C), preimmune sera (D), or anti-AKAP 220 antibody preabsorbed with 10 µg of purified AKAP 220 protein (E). F, a phase micrograph of TM4 cell is shown. Immune complexes were detected with fluorescein isothiocyanate-conjugated anti-rabbit secondary antiserum (A, C, D and E.) and Texas red-conjugated anti-goat secondary antiserum (B). Double label immunofluorescence of RII (A) and AKAP 220 (B) were analyzed in the same focal plane.
Anchoring of PKA at specific subcellular sites is thought to be a regulatory mechanism that permits the kinase access to certain substrates. Recently, it has become evident that AKAPs are a growing family of functionally related proteins which serve to localize the type II regulatory subunit (RII) of PKA to certain subcellular locations(29, 30) . Since there is little or no conservation in primary structure between AKAPs, the principle criterion used to identify these proteins is their ability to bind RII. Two predominant techniques are used: a solid-phase RII overlay assay and screening of bacterial expression libraries with RII as a probe(17) . Overlay techniques have shown that most cell types express 5-10 AKAPs(23, 31) , whereas other studies have demonstrated that hormonal stimulation or developmental signals promote the induction of certain anchoring proteins(15, 32) . Likewise, RII expression cloning techniques have been used to isolate several cDNAs and 8 AKAP sequences have been published to date (13, 14, 15, 22, 23, 33, 34) . One important property that has emerged from these cloning studies is that all AKAPs isolated by interaction cloning retain the ability to bind RII under the denaturing conditions used in the overlay assay.
The putative RII binding site of AKAP 220 is located in the
COOH-terminal third of the protein and probably includes residues
905-918. This sequence shares similarity with the RII binding
regions of AKAP 150, Ht 31, and MAP
2(14, 32, 34, 35) . In addition, a
recombinant fragment, including this region exhibits a similar affinity
for RII as the human thyroid anchoring protein, Ht
31(27) . Residues 905-918 are predicted to adopt a
helical confirmation and exhibit a partitioning of hydrophobic and
hydrophilic side chains when plotted on a helical wheel. All AKAPs seem
to possess an amphipathic helix region that forms the principle site of
contact with the RII dimer. Mutagenesis studies demonstrate that
disruption of secondary structure by the insertion of prolines
abolishes the interaction with RII(22, 27) . Other
evidence is that a peptide derived from residues 393-415 of Ht
31, which mimics the amphipathic helix, binds RII with nanomolar
affinity to block the AKAP interaction(23) . Since this peptide
blocks RII-AKAP 220 interactions, it suggests that AKAP 220 binds the
RII dimer in a manner similar to other AKAPs. AKAP 220 is, therefore, a
prototypic AKAP based on its ability to bind RII.
Another hallmark of AKAPs is their anomalous migration on SDS-PAGE gels. In fact, AKAP 75, AKAP 95, and MAP 2 all display apparent molecular weights which are approximately double their calculated molecular mass(14, 36, 37) . AKAP 220 follows this pattern as the calculated molecular weight from the cDNA sequence is 124,427, whereas the native protein migrates with a mobility of approximately 220 kDa on SDS-polyacrylamide gels. Potential explanations for this discrepancy in apparent molecular weight may be related to the numerous acidic residues in the amino-terminal portion of the protein or by phosphorylation or other post-translation modifications.
Although most AKAPs share some common RII binding characteristics, each anchoring protein is targeted to specific subcellular sites(6) . In recent years AKAPs have been identified that tether PKA to specific subcellular sites(13, 14, 15, 22, 23, 33, 34, 35, 36) . It appears that AKAP 220 may be targeted to peroxisomes through the last three residues of the protein, Cys-Arg-Leu. This sequence conforms to a peroxisomal targeting signal 1 motif(38, 39, 40) . Work originally performed on the COOH terminus of the firefly luciferase has demonstrated that the sequence Ser-Lys-Leu is necessary and sufficient for peroxisomal targeting(39) . Analysis of mammalian peroxisomal targeting motifs have determined that Leu is the only invariant residue in the triplet, whereas Ser can be replaced by Cys or Ala at the first position and Arg can be substituted for Lys at position two. Therefore, the Cys-Arg-Leu triplet in AKAP 220 is likely to facilitate peroxisomal localization of AKAP 220 and may represent its targeting domain(38, 39, 40) . Our immunocytochemical analysis supports this association of AKAP 220 with testicular peroxisomes. Future studies are planned to establish whether the Cys-Arg-Leu triplet is sufficient for the peroxisomal targeting of AKAP 220 and whether the anchoring protein is an integral component of the peroxisomal matrix.
Peroxisomes are small organelles present in all
cell types that function prominently in cellular lipid metabolism, such
as the -oxidation of fatty acids. These organelles have been
associated with cAMP-responsive events such as androgen
biosynthesis(41) . In particular, there appears to be a direct
correlation between testicular peroxisome volume and testosterone
secretion(42, 43) . However, the role of targeted PKA
in peroxisomes is currently unknown. At least two classes of
peroxisomes exist in most cells, these can be distinguished by catalase
staining methods(44) . Our double label immunofluorescence
experiments, using the 70-kDa peroxisomal marker, show that AKAP 220
overlaps with only a portion of peroxisomes. At this time we do not
know which subset of peroxisomes co-localize with AKAP 220; however,
the association of AKAP 220 with rat testicular peroxisomes may provide
a potential role for targeted PKA regulation of steroid biosynthesis.
AKAP 220 may also serve as an adaptor protein that co-localizes PKA with other signaling molecules. Until recently, AKAPs were thought to function exclusively in the localization of PKA. However, this view has been modified to include recent findings that demonstrate that AKAP 79 also binds the calcium/calmodulin-dependent phosphatase 2B, calcineurin(25) . Likewise, another AKAP, MAP 2, has been shown to co-purify with subcellular fractions that contain type 2A phosphatase (45) . Co-localization of kinases and phosphatases may account for the exquisite modulation of certain phosphorylation events that are necessary for maintaining cellular homeostasis(46) . In addition, proteins with the peroxisomal targeting signal have been shown to function as carrier proteins that piggy-back other molecules into peroxisomes(47) . Therefore, AKAP 220 has the potential to regulate peroxisomal functions through multiple mechanisms. Future studies are under way to define the role of AKAP 220 and associated proteins in testicular signaling events.