From the Veterans Affairs Medical Center and Oregon
Health Sciences University, Portland, Oregon 97201, the
¶ Department of Pharmacology, Kyoto University Faculty of
Medicine, Kyoto 606-8501, Japan, and the
Department of Cell
Biology, Vanderbilt University, Nashville, Tennessee 37232
Received for publication, December 13, 2000, and in revised form, January 25, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cAMP-dependent protein kinase
(PKA) is targeted to specific subcellular compartments through its
interaction with A-kinase anchoring
proteins (AKAPs). AKAPs contain an amphipathic helix domain
that binds to the type II regulatory subunit of PKA (RII). Synthetic
peptides containing this amphipathic helix domain bind to RII with high
affinity and competitively inhibit the binding of PKA with AKAPs.
Addition of these anchoring inhibitor peptides to spermatozoa inhibits
motility (Vijayaraghavan, S., Goueli, S. A., Davey, M. P.,
and Carr, D. W. (1997) J. Biol. Chem. 272, 4747-4752). However, inhibition of the PKA catalytic activity does not
mimic these peptides, suggesting that the peptides are disrupting the
interaction of AKAP(s) with proteins other than PKA. Using the yeast
two-hybrid system, we have now identified two sperm-specific human
proteins that interact with the amphipathic helix region of AKAP110.
These proteins, ropporin (a protein previously shown to interact with
the Rho signaling pathway) and AKAP-associated sperm protein, are 39%
identical to each other and share a strong sequence similarity with the
conserved domain on the N terminus of RII that is involved in
dimerization and AKAP binding. Mutation of conserved residues in
ropporin or RII prevents binding to AKAP110. These data suggest that
sperm contains several proteins that bind to AKAPs in a manner similar
to RII and imply that AKAPs may have additional and perhaps unique
functions in spermatozoa.
PKA1 is a ubiquitous,
multifunctional kinase involved in the regulation of a diverse array of
cellular events. The PKA holoenzyme consists of four subunits, two
catalytic and two regulatory. The regulatory subunits form dimers
through an interaction at the N terminus whereas the C terminus
contains two tandem repeat sequences, which form the cAMP binding
sites. Binding of cAMP to the regulatory subunits promotes the
dissociation and activation of the catalytic subunits. A second
function of the regulatory subunits is to target or anchor PKA to
specific subcellular locations within the cell.
A major advance in signal transduction research in recent years is the
understanding that the actions of many signaling molecules are
spatially restricted and coordinated through cell- and
function-specific targeting of these enzymes and their substrates (1).
PKA is anchored to specific cellular compartments through the
interaction of the regulatory subunit with a family of proteins
referred to as A-kinase anchoring
proteins (AKAPs) (reviewed in Refs. 2-4). Numerous AKAPs
have been cloned and biochemically characterized. Several AKAPs have
been shown to simultaneously bind to PKA and other signal transduction
molecules such as calmodulin, protein phosphatase 1 (PP1), calcineurin
(PP2B), and protein kinase C (5-9). This has led to a model in which
AKAPs act as scaffolding molecules that coordinate the actions of
several kinases and phosphatases all located within one cellular compartment.
The structural feature of AKAPs that promotes interaction with PKA has
been known for some time (10). AKAPs contain an amphipathic helix
region and bind to the type II regulatory subunit of PKA via the
hydrophobic face. The identification of this binding domain has
facilitated the design of reagents that have been used to determine
experimentally the physiological consequences of the interaction of PKA
with AKAPs. Synthetic peptides encompassing the amphipathic helix
binding domain are potent competitive inhibitors of PKA·AKAP
interaction (11) and therefore are referred to as anchoring
inhibitor peptides (AIPs). Addition of AIPs to a variety of somatic
cells inhibits PKA modulation of cellular events. For example,
microinjection of AIPs into hippocampal neurons causes a
time-dependent decrease in AMPA/kainate-responsive currents whereas control peptides had no effect on channel activity, suggesting that PKA anchoring is required for PKA modulation of the AMPA/kainate channels (12). Since this initial finding, several laboratories have
used AIPs to demonstrate that anchoring is required for PKA modulation
of L-type Ca2+ channels (13), calcium-activated
potassium channels (14), insulin secretion from pancreatic beta cells
(15), and phosphatidylinositide turnover in myometrial cells (16, 17).
In all of the above examples, the action of AIPs is mimicked by the
addition of reagents that inhibit the catalytic activity of PKA. These
data support a model where AKAPs interact with the PKA regulatory
subunit to anchor or target the catalytic subunit to relevant
physiological substrates.
Several sperm AKAPs have been identified and characterized (18-27).
The most prominent AKAP detected by RII overlay assay of bovine, human,
mouse, and monkey spermatozoa is AKAP110 (20, 21, 27). Northern
analysis suggests that AKAP110 is expressed only in spermatozoa, and
immunofluorescence studies detecting AKAP110 in the flagella suggest
that this protein may be involved in regulating motility. Addition of
S-Ht31 (stearated-Ht31 is a cell permeable AIP) to bovine caudal
epididymal spermatozoa inhibits motility in a time- and
concentration-dependent manner (26). A control peptide,
S-Ht31-P, identical to S-Ht31 except for an isoleucine to proline
substitution that prevents amphipathic helix formation, had no effect
on motility. Surprisingly, inhibition of PKA catalytic activity by
addition of H-89 or S-PKI had little effect on basal motility or
motility stimulated by agents previously thought to work via PKA
activation (26). These data suggest that proteins interacting with
sperm AKAPs regulate motility in a manner that is independent of PKA
catalytic activity. These results have been confirmed by a different
approach. McKnight and colleagues (28), using mutant mice lacking
RII One hypothesis consistent with the above data is that sperm AKAPs are
interacting with proteins other than RII via the amphipathic helix
domain. This would explain how AIPs could be regulating motility in a
manner independent of both the regulatory and catalytic subunits of
PKA. Using the yeast two-hybrid system to screen a human testis
library, we have now identified several sperm-specific proteins that
bind to fragments of AKAP110 containing the amphipathic helix domain.
Binding studies demonstrate that these proteins bind to AKAP110 in a
manner homologous with RII. Based on these data, we propose a model for
the function of AKAPs in spermatozoa that is very different than the
model for AKAPs in somatic cells.
Yeast Two-hybrid Screening--
The yeast strain
EGY48(p2op-lacZ) containing constructs of pLexA human AKAP110,
truncated hAKAP110-(1-350), and human ropporin was transformed with a
pB42AD-fused human testis cDNA library following protocols for the
Matchmaker LexA Two-Hybrid System (CLONTECH).
Approximately 2 × 108 transformants were screened
with each construct on 150-mm plates containing 5 × 104 clones with SD/Gal/Raf/-His/-Trp/-Ura/-Leu/+Xgal + BU
salts. Blue colonies, positive for Purification of Recombinant Proteins--
Ropporin and mutant
ropporin (L18A) were expressed as pET30a N-terminal
His6-tagged fusion proteins in Escherichia coli
(BL21(DE3)) and were purified by fast protein liquid chromatography
using Hi-Trap chelating Sepharose columns (Amersham Pharmacia Biotech). One liter of LB broth + 30 µg/ml kanamycin was inoculated and grown
to mid-logarithm phase before adding 1 mM
isopropyl-1-thio- Plasmids--
Plasmids for expression of recombinant hAKAP110,
hAKAP110-(1-350), mRII Northern Blot Analysis of Human ASP--
A Mouse Multiple Tissue
Northern blot (CLONTECH), containing 2 µg of
poly(A)+ RNA per lane, was screened using the full-length
ASP fragment generated in the plasmids section. The probe was
32P-labeled using the High Prime DNA labeling kit (Roche
Molecular Biochemicals). Hybridization of the probe was carried out at
42 °C for 18 h in Ullrich's buffer/2% SDS. The blot was
washed at room temperature 2× in SSC, 2× in SSC/2% SDS at 65 °C
and finally in 0.1% SSC at room temperature. An exposure of the blot
was then made on x-ray film for 72 h.
In Vitro Binding Assay of PKA Regulatory Subunit (RII
Prior to the addition of purified proteins, 200 µl of the
GST-hAKAP110-(1-350) was incubated with 750 µl of Blotto and 10 mM dithiothreitol for 30 min at room temperature. 1 µg of
RII PCR and Sequencing--
PCR was done directly on diethyl
pyrocarbonate-H2O suspensions of yeast cell
positives using vector-specific primers flanking the multiple cloning
site of the pB42AD vector (29, 30). The resulting PCR products were
gel-purified, and sequence analysis was performed using the Big-Dye
Terminator Sequencing Kit (Applied Biosystems, Foster City, CA) and the
same vector specific primers. The Y-Der yeast DNA extraction kit
(Pierce, Rockford, IL) was used to recover plasmid DNA from positives
that wouldn't amplify using the above method. Analysis of sequence
data, sequence comparison, and alignments was performed using the
MacVector ClustalW program (Oxford Molecular Group) and the BLAST
program (31) provided by the NCBI server at the National Library of
Medicine/National Institutes of Health.
Rapid Amplification of 5'-cDNA Ends (5'-RACE)--
The
5'-RACE was performed using a Marathon cDNA amplification kit and
Human Testis Marathon-Ready cDNA (CLONTECH) as
described with the accompanying procedures. The primers used to obtain
full-length PCR products were previously stated in the plasmids section.
Site-directed Mutagenesis--
Plasmid constructs pET30a mouse
ropporin, pET11d mouse RII
The resulting mutated DNA was transformed into E. coli Super
Competent JM109 cells (Promega, Madison, WI) and grown on
antibiotic-resistant LB Agar plates. The mutations were verified by
sequence analysis using vector-specific primers.
Immunocytochemistry--
Adult male mice were sacrificed by
CO2 asphyxiation, and a sperm suspension was obtained by
mincing the cauda epididymides in buffered saline (145 mM
NaCl and 5 mM Hepes, pH 7.4). Sperm were then fixed 30 min
in 4% formaldehyde in 0.1 M sodium phosphate buffer, pH
7.4), attached to coverslips, permeabilized in absolute acetone for 10 min at Yeast Two-hybrid Screening of Human Testis cDNA
Library--
AKAP110 is a sperm-specific AKAP that binds to the type
II regulatory subunit of PKA via an amphipathic helix-binding motif located at amino acid position 124-143 (27). To determine if other
proteins also bind to AKAP110 via the amphipathic helix binding domain,
a human testis cDNA library was screened using an N-terminal
fragment of AKAP110-(1-350) as bait in a yeast two-hybrid procedure.
Positives were then co-transformed in the two-hybrid system with
another fragment of AKAP110-(349-660) that does not contain an
amphipathic helix domain. Three positives were identified that bound to
the amphipathic helix-containing fragment (1) but not to the other
fragment (349) (Fig. 1). The pB42AD
plasmids were recovered from these clones, and the cDNA inserts
(1.4, 1.3, and 1.1 kb in size) were sequenced.
Sequence Analysis of AKAP110 Binding
Proteins--
Sequence analysis demonstrated that the 1.4-kb insert
encodes for the human type II regulatory subunit of the
cAMP-dependent protein kinase, RII
The sequence obtained from the 1.3-kb insert is 94% identical to the
amino acid sequence of the murine protein ropporin (GenBankTM accession number AF178531), which we previously isolated as a
binding partner of a Rho effector, rhophilin (32). The human ropporin
(h-ropporin) sequence has been submitted to the GenBankTM data base
(accession number AF231410). Ropporin is a sperm-specific protein
localized in the principal piece and the end piece of sperm flagella
(32). Ropporin forms homodimers and binds to rhophilin, and both
proteins have been shown to co-precipitate in vitro with Rho
(32). Once again, in addition to sequence that was homologous to the
coding region for m-ropporin, the pB42AD insert contained sequence
upstream (165 bases) of the ropporin start site, adding 55 amino acids
of 5'-untranslated repeat as a bridge between the pB42AD fusion protein
and the h-ropporin.
The sequence obtained from the 1.1-kb insert is a novel protein. It is
39% identical to h-ropporin but does not match any other protein in
the GenBankTM data base and will hereafter be referred to as
AKAP-associated sperm protein or
ASP. ASP also contains a 5'-untranslated repeat bridge (57 bases, 19 amino acids) between the vector and the start site. This sequence has
been submitted to the GenBankTM data base (accession number AF239723). The optimal alignment (MacVector ClustalW Alignment Program) of m-ropporin, h-ropporin, and ASP is shown in Fig.
2. ASP is highly homologous with the
N-terminal 80 residues of ropporin and only moderately homologous with
the rest of the molecule, suggesting the N terminus contains a
conserved domain that may have an important function in
spermatozoa.
Tissue Distribution of ASP mRNA--
We have previously shown
that m-ropporin is detected only in the testis and then only in the
most inner part of the seminiferous tubules region, suggesting this
protein is expressed in developing spermatozoa (32). To determine the
tissue distribution of ASP, Northern blots containing 2 µg of
poly(A)+ RNA from eight different adult mouse tissues were
probed with 32P-labeled ASP cDNA. A single message was
detected only in the testis (Fig. 3),
suggesting this protein is testis-specific and possibly sperm-specific.
Using a linear regression analysis of a plot of the log10
versus the RF of the molecular weight markers, the ASP mRNA was calculated to be ~1.05 kb. This was bigger than expected based on the insert size (850 bases) from the
yeast vector. To determine if the insert represented full-length cDNA for ASP, 5'-RACE was performed using Human Testis
Marathon-Ready cDNA (CLONTECH) as template. An
additional 144 bases were identified using this technique, bringing the
total number of bases to 994 or approximately the same size as the
calculated mRNA. Although this new region contains an open reading
frame continuous with the rest of the protein, it does not contain an
alternate start site. At the time of submission of this manuscript, a
BLAST search of the human genome data base does not detect either human
ropporin or ASP.
Alignment of Sperm Proteins with RII--
As mentioned above, the
N-terminal regions of ropporin and ASP are the most conserved.
The sequence in this region is also similar to the N terminus of the
type II regulatory subunit of PKA and two other sperm-specific
proteins, SP-17 (GenBankTM accession number Q15506) (33,
34) and fibrousheathin II (FSII)
(GenBankTM accession number
NM_012189).2 Optimal
alignment (MacVector ClustalW alignment program) of RII
The N-terminal 44 amino acids of RII contain the domains responsible
for homodimerization and binding to AKAPs (Fig. 4B). The
sequence identity of h-RII Functional Comparison of RII Colocalization of AKAP110 and Ropporin in Mouse
Spermatozoa--
Cauda epididymal mouse spermatozoa immunostained with
antibodies to AKAP110 exhibited specific staining of both the principal piece segment and of the dorsal margin of the acrosomal segment (Fig.
6, A and B);
specific staining was not detectable in other sperm segments. Cauda
epididymal spermatozoa immunostained with anti-ropporin exhibited
specific staining of the principal piece segment and as well as of the
cytoplasmic droplet located at the distal end of the midpiece (Fig. 6,
C and D). Control samples exposed to non-immune
rabbit IgG exhibited no detectable fluorescence (not shown). These data
indicate that AKAP110 and ropporin are both located in the principal
piece. In addition, each protein appears to uniquely occupy another
compartment, AKAP110 in the acrosome and ropporin in the cytoplasmic
droplet and midpiece.
In somatic cells, experimental data suggest that AKAPs anchor or
target PKA to key physiological substrates. Disruption of PKA·AKAP
interaction in a cell results in the loss of cAMP/PKA modulation of
specific events. For example, oxytocin stimulates uterine smooth muscle
contraction by increasing phosphatidylinositide turnover, which in turn
promotes the release of intracellular calcium. Addition of cAMP to
myometrial cells inhibits the action of oxytocin by stimulating PKA
activity. However, cAMP does not inhibit oxytocin if the myometrial
cells have been pretreated with anchoring inhibitor peptides (AIPs) or
with PKA inhibitors such as H-89 (17). These data support a model in
which AKAPs interact with the PKA regulatory subunit to anchor the
catalytic subunit to a location within the cells where it is available
to phosphorylate the appropriate substrate if and when it becomes activated.
Although AIPs are potent inhibitors of bovine sperm motility (26),
several lines of evidence suggest that the function of AKAPs in sperm
is different from somatic cells. First, PKA inhibitors such as H-89 or
S-PKI have little or no effect on bovine sperm motility (26), and
second, altering the subcellular location of the PKA catalytic subunit
using targeted gene disruption of the type II regulatory subunit does
not significantly effect mouse sperm motility (28). These data support
our hypothesis that sperm AKAPs are interacting with proteins other
than PKA. To be consistent with the above data, this new interaction
would have to involve the amphipathic helix domain of the AKAPs and
therefore be subject to disruption by the addition of AIPs. To
determine if sperm AKAPs interacted with proteins other than RII, a
human testis library was screened using a fragment of AKAP110 that
contained the amphipathic helix domain. Three proteins were identified
that bound to this fragment, but not other fragments, of AKAP110. One protein was the regulatory subunit of PKA, RII Both ropporin and ASP share strong sequence similarity with the AKAP
binding domain of RII The main function of somatic cell AKAPs is to anchor or target the PKA
catalytic subunit through an interaction with the regulatory subunit.
No other somatic cell proteins have yet been identified that would
compete with RII for binding to AKAPs. In contrast, sperm AKAPs can
bind RII, but their ability to regulate sperm functions such as
motility and the acrosome reaction appears to be independent of the
location of the PKA catalytic subunit. In addition, all of the sperm
RII homologs could potentially compete with RII, or each other, for
binding to sperm AKAPs. These data suggest that sperm AKAPs may have
additional and perhaps unique functions compared with somatic cells.
Although a more exhaustive study will need to be performed, present
data suggest that spermatozoa are the only cells that contain proteins,
other than RII, that interact with the amphipathic helix domain of
AKAPs. The fact that sperm expresses at least four of these proteins
indicates they have evolved a unique and important use for this
interaction. It is possible that the different AKAP-binding proteins
might act on different pathways. Several AKAP-binding proteins (RII One possible role of sperm AKAPs might be to function as a scaffolding
protein for the Rho-GTPase pathway. AKAP110 and ropporin co-localize in
the sperm flagellum. Ropporin was originally identified in a yeast
two-hybrid screen of a mouse testis library using rhophilin as bait
(32). Rhophilin serves as an adaptor protein capable of binding both
ropporin and Rho simultaneously. Rho is a small GTPase that functions
as a molecular switch, regulating various cellular processes such as
cell adhesion, gene expression and cytokinesis, smooth muscle
contraction, and motility (38). Rho has also been shown to regulate
protein phosphatases through activation of Rho-kinase (39). Regulation
of myosin phosphatase by Rho and Rho-kinase controls smooth muscle
contraction and actin/myosin interaction in non-muscle cells (40). We
hypothesize a similar pathway may exist in sperm. Evidence supporting
this hypothesis includes: 1) inhibition of Rho by ADP ribosylation
using C3 exoenzyme inhibits bovine sperm motility (41); and 2) addition
of AIPs to spermatozoa produces a 2-fold increase in PP1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, have shown that the catalytic subunit is no longer located
along the flagellum but instead is concentrated in the cytoplasmic
droplet, yet the spermatozoa are motile and the mice are fertile. These
data suggest that neither RII
nor RII
-dependent
localization of PKA catalytic subunit is necessary to support motility.
Thus, it appears that AIPs may be exerting an effect on spermatozoa
that is independent of AKAP·RII
interaction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity, were
re-streaked on SD/-His/-Trp/-Ura plates, then replated on
SD/Gal/Raf/-His/-Trp/-Ura/-Leu/+X-gal + BU salts to re-test for
activation of both the lacZ and LEU2 reporter genes. Double
positives were then plated on master plates containing
SD/-His/-Trp/-Ura and used for yeast PCR and sequence analysis.
-D-galactopyranoside to induce protein
expression at 37 °C for 2 h. The cells were then pelleted by
centrifugation at 2000 × g for 20 min, sonicated in
buffer A on ice (20 mM HEPES, 500 mM NaCl, 20 mM imidazole, 1 mM
4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride, pH 7.4), and
clarified by centrifugation at 15,000 × g for 20 min at 4 °C, and the resultant supernatant was passed through a
0.45-µm filter. The supernatant was then applied to the
Ni2+-charged Hi-Trap chelating column. Bound proteins were
eluted with a stepwise gradient of imidazole (0-0.5 M)
in buffer A. Fractions containing purified protein were identified by
Coomassie Blue staining of 10% SDS-polyacrylamide gel electrophoresis
gels. pET11d-RII
and mutant RII
(L14A) were expressed in E. coli (BL21(DE3)) and purified by cAMP-agarose affinity column as
previously described (10).
, h-ropporin, hASP, hSP17, and hFSII in a
yeast two-hybrid system, as histidine-tagged or GST fusion proteins in
E. coli were prepared as follows. The pET30a hAKAP110
full-length construct and its truncated form hAKAP110-(1-350) (27)
were digested with EcoRI and XhoI, gel-purified,
and ligated into pLexA, pB42AD (CLONTECH, Palo
Alto, CA), and pGEX5x-1 (Amersham Pharmacia Biotech, Piscataway, NJ)
vectors cut with the same restriction enzymes. PCR was performed on
pET11d RII
with forward primer
5'-CCGGAATTCATGAGCCACATCCAGATCCCG-3' and reverse primer
5'-CCGCTCGAGCACACTGAGAAGGCTCCAAGATTC-3', respectively, containing an
EcoRI and XhoI restriction site. The PCR product was digested with EcoRI/XhoI, gel-purified, and
ligated into pLexA and pB42AD. Full-length PCR products of ropporin,
SP17, and FSII were obtained by 5'-RACE of a Marathon Ready Human
Testis cDNA library (CLONTECH). Ropporin
forward primer 5'-GGATTCATGGCTCAGACAGATAAGCCAACATG-3' and
reverse primer 5'-CCCTCGAGAATTGTGCTGTTACTCCAGCCAAACC-3', SP17 forward
primer 5'-AGATCCATGTCGATCCATTCTCCAACACCCA-3' and reverse primer
5'-ATTTGCGGCCGCTGGAGGTAAAACCAGTGTCCTCACTTG-3', FSII forward primer
5'-AGATCCATGTCGATCCATTCTCCAACACCCAC-3' and reverse primer 5'-ATTTGCGGCCGCTGGAGGTAAAACCAGTCTCCTCACTTG-3'. Ropporin had
EcoRI/XhoI restriction sites added to the
primers, SP17 and FSII primers had BamHI/NotI
restriction sites added. Ropporin was digested and ligated into pET30a,
pLexA, and pB42AD; SP17 and FSII were subcloned into pLexA. ASP
was obtained using the yeast two-hybrid system and Human Testis
Matchmaker LexA cDNA (CLONTECH),
EcoRI/XhoI-digested, and ligated into pLexA
and pET30a.
) and
Ropporin to AKAP110--
E. coli transformed with pGEX-5X-1
plasmid encoding human AKAP110-(1-350) or AKAP110-P (containing a
proline substitution for leucine 131) were grown to mid-logarithm phase
at 37 °C in 1 liter of LB medium. They were cultured for an
additional 2 h at 37 °C in the presence of 0.2 mM
isopropyl-
-D-thiogalactopyranoside to induce synthesis
of the fusion protein. Crude extracts were prepared by sonicating the
bacteria in 20 mM Tris-HCl, pH 8.0, 100 mM
NaCl, and 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride. 2% Tween 20 (v/v) was added to the extract and rotated
at room temperature for 1 h. The extract was centrifuged at
15,000 × g for 20 min before passing the supernatant
through a 0.45-µm filter. The supernatant was incubated for 30 min at room temperature with 3 ml of glutathione-Sepharose before extensive washing in phosphate-buffered saline to remove nonspecifically bound proteins.
, RII
(L14A), 3 µg of ropporin and 4 µg of ropporin (L18A)
were added to separate tubes containing the Blotto/AKAP110 mix and rotated at room temperature for 2 h. After washing extensively with phosphate-buffered saline and 10 mM dithiothreitol,
proteins were eluted by boiling in SDS gel-loading buffer and separated by 10% SDS-polyacrylamide gel electrophoresis. Regulatory subunit RII
was detected by Western blotting using rabbit antisera against RII
(6825) and secondary anti-rabbit horseradish peroxidase
conjugate (Sigma Chemical Co., St. Louis, MO). Ropporin was detected by conjugated horseradish peroxidase-Anti-S-Protein (Novagen, Madison, WI).
, and pGEX5x-1 containing
hAKAP110-(1-350) were used as PCR templates for the QuikChange 1-day
site-directed mutagenesis method (Stratagene, La Jolla, CA). Aligned
leucines (see Fig. 4A below) at positions 18 and 14 of ropporin and RII
, respectively, were mutated to alanines using
the following primers: Ropporin (L18A), forward primer
5'-TGCCGGAATTGGCAAAGCAGTTTAC-3', reverse primer
5'-GTAAACTGCTTTGCCAATTCCGGCA-3'; RII
(L14A), forward primer
5'-TCACGGAGCTGGCACAGGGCTACA-3', reverse primer
5'-TGTAGCCCTGTGCCAGCTCCGTGA-3'. Leucine 131 within the amphipathic helix region of hAKAP110 was mutated to proline using the
following primers: forward primer 5'-ATGCTAACCGCCCAACGAATCTAG-3', reverse primer 5'-CTAGATTCGTTGGGCGGTTAGCAT-3'.
20 °C and air-dried. For immunostaining, cells were first
incubated 1 h in blocking buffer of Tris-saline (TN = 150 mM NaCl, 25 mM Tris-HCl, pH 8.0, and 0.05%
Tween 20) containing 2.5% bovine serum albumin and 5% goat
serum and then successively incubated in primary and secondary
antibodies also diluted in blocking solution. Between all incubation
steps, coverslips were washed three times in TN containing 1% goat
serum. Primary antibodies, prepared in rabbits, included
affinity-purified IgG to AKAP110 and ropporin. Control samples
substituted identical levels of affinity-purified non-immune rabbit IgG
for immune IgG. Cy3-conjugated affinity-purified secondary antibodies
were obtained from Jackson ImmunoResearch (West Grove, PA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (62K):
[in a new window]
Fig. 1.
Fragments of AKAP110 containing the
amphipathic helix binds RII, ropporin, and ASP. A human testis
library was screened using AKAP110-(1-350) as bait. Positives included
RII , ropporin, and AKAP-associated sperm protein (ASP).
These positives were then co-transformed with a fragment of
AKAP110-(349-660) that does not contain the amphipathic helix. The
transformants were plated on a selective medium and subjected to
-galactosidase assay.
(GenBankTM accession
number NM004147). The first base of the insert lines up with
base 142 of the deposited sequence, which lists the coding region as
190-1404. Thus, the 1.4-kb insert contains the full-length coding
region for PKA RII
protein plus an additional 16 amino acids from
the 5'-untranslated region. Although this additional region would
normally not be translated, there are no stop codons within this
segment, and thus these additional amino acids functioned as a bridge
between the pB42AD fusion partner and RII
.
View larger version (60K):
[in a new window]
Fig. 2.
Amino acid sequence homology of mouse
ropporin, human ropporin, and AKAP-associated sperm protein
(ASP). Identical amino acids shared by more than
one protein are shaded dark gray. Functionally similar amino
acids are shaded light gray.
View larger version (38K):
[in a new window]
Fig. 3.
Northern blot analysis of tissue distribution
of ASP mRNA expression. A Northern blot containing 2 µg of
poly(A)+ RNA per lane was probed with
32P-labeled ASP cDNA and detected by autoradiogram. The
tissue source of the RNA is indicated at the top of each
lane, and the positions of the molecular size markers (bp × 10 3) are shown on the left.
and RII
(residues 1-46 or 45, respectively, of both human and mouse) with the
corresponding regions of ropporin, ASP, SP17, and FSII is shown in Fig.
4A. The dark gray
shading indicates sequence identity, and the light gray
shading indicates sequence similarity. Although these sperm
proteins share high sequence similarity with the N-terminal region of
RII, they have little or no homology with other regions of RII such as
the nucleotide binding domains. The only other protein identifiable by
sequence homology, sharing the characteristics of having an AKAP
binding domain but not a cyclic-nucleotide binding domain, is a
hypothetical protein from Caenorhabditis elegans F39H12.3.
The location of this protein in C. elegans is still
undetermined.
View larger version (34K):
[in a new window]
Fig. 4.
Sequence alignment of the AKAP binding domain
of RII with ropporin, ASP, SP17, FSII, and an unknown C. elegans protein. A, the N terminus of
RII , RII
, ropporin, ASP, SP17, FSII, and a C. elegans
protein were aligned using the MacVector ClustalW software program.
Identical amino acids shared by more than five proteins are
shaded dark gray. Functionally similar amino acids are
shaded light gray. The percent sequence identity of each
protein compared with human RII
is shown in the right-hand
column. B, the location of the AKAP binding,
dimerization, and cAMP binding domains of RII
are shown.
, h-ropporin, hASP, hSP17, hFSII, and
hRI
with hRII
(1-44) is 70, 30, 32, 45, 34, and 18%,
respectively. It is interesting to note that all of these newly
identified AKAP-binding proteins have a higher sequence homology with
RII than RI does with RII (RI is not even picked up in the
position-specific iterated BLAST search), suggesting that all of
these proteins may bind AKAPs with a higher affinity than RI. The
positions that are most conserved within this region are those that
have been identified as being key residues involved in either
homodimerization or AKAP binding (35-37). For example, Li and Ruben
(36) showed that substitution of Ala for Leu-13 or Phe-36 in RII
generates monomeric RII
that cannot bind to AKAPs. Both of these
residues are 100% conserved in all of the above proteins.
with Ropporin--
In
vitro binding assays were performed to determine if ropporin
interacts with AKAPs in a manner functionally homologous with RII
.
As mentioned above, mutation of Leu-13 (Leu-13 in RII
is equivalent to Leu-14 in RII
) to Ala inhibits RII binding to AKAPs (36). To confirm these results, a pull-down assay using GST-AKAP110 was
performed. Wild type RII
bound to AKAP110, but the RII
alanine mutant did not, even in the presence of excess mutant protein (Fig.
5A). A comparable mutant was
made for ropporin, substituting Ala for Leu-18 (see alignment in Fig.
4A). As with RII
, only the wild type ropporin bound to
AKAP110 (Fig. 5B). One characteristic of RII
·AKAP
interaction is that disruption of the secondary structure of the
amphipathic helix region, by insertion of a proline residue, disrupts
binding. Incubation of either RII
or ropporin (wild type or Ala
mutant) with AKAP110-P (proline substituted for leucine at position
130) did not produce any detectable interaction.
View larger version (47K):
[in a new window]
Fig. 5.
In vitro binding of wild type and
mutant RII and ropporin to immobilized wild
type and mutant AKAP110. An equal amount of AKAP110-(1-350) or
AKAP110-P (1-350 with a proline for leucine substitution at position
131) was conjugated to glutathione beads (AKAP110 with
arrows indicates Coomassie Blue stain of AKAP110 or
AKAP110-P immobilized on each column). A, cell lysates
containing wild type RII
or mutant RII
(ALA,
indicating a substitution of alanine for leucine 14) or ropporin
(B) (WT or alanine for leucine 18) were incubated
with the AKAP110 or AKAP110-P beads and then washed. Bound proteins
were precipitated and subjected to Western blot analysis of total
lysates (Load, left panel), AKAP110 pull-down
(middle panel), or AKAP110-P pull-down (right
panel). The positions of RII and ropporin are shown by
arrows.
View larger version (59K):
[in a new window]
Fig. 6.
Colocalization of AKAP110 and ropporin in
sperm flagellum. Matched phase contrast (A and
C) and fluorescence images (B and D)
of cauda epididymal spermatozoa immunostained with anti-AKAP110
(A and B) and anti-ropporin (C and
D). Both antibodies give positive staining of the principal
piece segment of the flagellum (pp). Positive staining of
the dorsal surface of the acrosome (a) is detected with
anti-AKAP110, and positive staining of the cytoplasmic droplet
(cd) is noted with the anti-ropporin antibody.
mp, midpiece segment. All photographs were taken at the same
magnification, and the bar in A and C
represents 10 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
. A second protein was
the human homolog of mouse ropporin (32), and the third was a novel
protein with 39% sequence similarity to ropporin. We have named the
novel protein AKAP-associated sperm protein or ASP.
, suggesting that, like RII
, they interact
with the amphipathic helix region of AKAPs. Using BLAST position-specific iterated searches, two other sperm proteins, SP17 and fibrosheathin II, have also been identified as having sequence
similarity with RII in this region, suggesting spermatozoa contain at
least four proteins capable of binding the amphipathic helix region of
AKAPs. These other proteins do not contain consensus motifs that would
indicate they bind to either cyclic nucleotides or the catalytic
subunit of PKA. The amino acids that are most conserved between RII and
these other four proteins are the same amino acids that have been
identified as being important for RII dimerization and RII·AKAP
interaction (35, 36). Site-directed substitution of alanine for leucine
at position 14 in RII
or the equivalent position in ropporin
disrupted the interaction of these proteins with AKAP110. Likewise,
proline substitution at position 132 within the amphipathic helix
region of AKAP110 disrupted interaction with both RII
and ropporin.
These data suggest that ropporin and the other molecules are functional
homologs of RII
in their ability to bind to AKAPs. Thus, it appears
that the classic model of AKAP function in somatic cells is
fundamentally different in spermatozoa.
, ropporin, SP17, and FSII) are located in the flagellum, suggesting any
or all of them may be involved in regulating motility. Both AKAP110 and
SP17 have been detected in the acrosomal region of the head, implying
these proteins may be involved in regulating head associated functions
such as the acrosome reaction or sperm/egg binding.
2 activity
and inhibits motility.3
PP1
2, a unique sperm serine/threonine phosphatase, is a key biochemical factor regulating motility (42, 43). Several AKAPs have
been shown to interact with phosphatases (44), including AKAP220 (9),
which is expressed in spermatozoa (25) and shares a 33% sequence
similarity with AKAKP110. These observations suggest that Rho may
regulate sperm motility via a mechanism similar to the one controlling
smooth muscle contraction and that AKAP110 or other sperm AKAPs may
function as scaffolding molecules controlling the location of Rho,
rhophilin, ropporin, and possibly Rho-kinase and PP1. Further
experiments will be needed to confirm the precise role of sperm AKAPs
and the Rho-GTPase pathway in regulating sperm motility.
![]() |
FOOTNOTES |
---|
* This research was supported by National Institutes of Health Grants HD36408 (to D. W. C.) and HD20419 (to G. E. O.) and by a grant-in-aid for specially promoted research from the Ministry of Education, Culture, Science and Sports of Japan (to S. N.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF231410 and AF239723.
§ To whom correspondence should be addressed: Veterans Administration Medical Center, R&D-8, 3710 SW Veterans Hospital Rd., Portland, OR 97201. Tel.: 503-721-7918; Fax, 503-721-1082; E-mail: carrd@ohsu.edu.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M011252200
2 A. Mandal, M. J. Wolkowicz, S. Naaby-Hansen, and J. C. Herr, unpublished data.
3 S. Vijayaraghavan and D. W. Carr, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; AKAPs, A-kinase anchoring proteins; RII, type II regulatory subunit of PKA; AIPs, anchoring inhibitor peptides; ASP, AKAP-associated sperm protein; FSII, fibrosheathin II; SP17, sperm protein 17; PKI, PKA inhibitor peptide; GST, glutathione S-transferase; RACE, rapid amplification of cDNA ends; kb, kilobase(s); PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Pawson, T.,
and Scott, J. D.
(1997)
Science
278,
2075-2080 |
2. |
Dell'Acqua, M. L.,
and Scott, J. D.
(1997)
J. Biol. Chem.
272,
12881-12884 |
3. | Rubin, C. S. (1994) Biochim. Biophys. Acta 1224, 467-479[Medline] [Order article via Infotrieve] |
4. |
Scott, J. D.,
and Carr, D. W.
(1992)
News Physiol. Sci.
7,
143-148 |
5. | Coghlan, V. M., Perrino, B. A., Howard, M., Langeberg, L. K., Hicks, J. B., Gallatin, W. M., and Scott, J. D. (1995) Science 267, 108-111[Medline] [Order article via Infotrieve] |
6. | Klauck, T. M., Faux, M. C., Labudda, K., Langeberg, L. K., Jaken, S., and Scott, J. D. (1996) Science 271, 1589-1592[Abstract] |
7. | Nauert, J. B., Klauck, T. M., Langeberg, L. K., and Scott, J. D. (1997) Curr. Biol. 7, 52-62[Medline] [Order article via Infotrieve] |
8. |
Sarkar, D.,
Erlichman, J.,
and Rubin, C. S.
(1984)
J. Biol. Chem.
259,
9840-9846 |
9. | Schillace, R. V., and Scott, J. D. (1999) Curr. Biol. 9, 321-324[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Carr, D. W.,
Stofko-Hahn, R. E.,
Fraser, I. D.,
Bishop, S. M.,
Acott, T. S.,
Brennan, R. G.,
and Scott, J. D.
(1991)
J. Biol. Chem.
266,
14188-14192 |
11. |
Carr, D. W.,
Hausken, Z. E.,
Fraser, I. D.,
Stofko-Hahn, R. E.,
and Scott, J. D.
(1992)
J. Biol. Chem.
267,
13376-13382 |
12. | Rosenmund, C., Carr, D. W., Bergeson, S. E., Nilaver, G., Scott, J. D., and Westbrook, G. L. (1994) Nature 368, 853-856[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Johnson, B. D.,
Scheuer, T.,
and Catterall, W. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11492-11496 |
14. |
Wang, Z. W.,
and Kotlikoff, M. I.
(1996)
Am. J. Physiol.
271,
L100-L105 |
15. |
Lester, L. B.,
Langeberg, L. K.,
and Scott, J. D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14942-14947 |
16. |
Dodge, K. L.,
Carr, D. W.,
Yue, C.,
and Sanborn, B. M.
(1999)
Mol. Endocrinol.
13,
1977-1987 |
17. |
Dodge, K. L.,
Carr, D. W.,
and Sanborn, B. M.
(1999)
Endocrinology
140,
5165-5170 |
18. | Carrera, A., Gerton, G. L., and Moss, S. B. (1994) Dev. Biol. 165, 272-284[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Erlichman, J.,
Gutierrez-Juarez, R.,
Zucker, S.,
Mei, X.,
and Orr, G. A.
(1999)
Eur. J. Biochem.
263,
797-805 |
20. |
Horowitz, J. A.,
Wasco, W.,
Leiser, M.,
and Orr, G. A.
(1988)
J. Biol. Chem.
263,
2098-2104 |
21. |
Lin, R. Y.,
Moss, S. B.,
and Rubin, C. S.
(1995)
J. Biol. Chem.
270,
27804 |
22. | Mei, X., Singh, I. S., Erlichman, J., and Orr, G. A. (1997) Eur. J. Biochem. 246, 425-432[Abstract] |
23. |
Miki, K.,
and Eddy, E. M.
(1999)
J. Biol. Chem.
274,
29057-29062 |
24. | Pariset, C., and Weinman, S. (1994) Mol. Reprod. Dev. 39, 415-422[Medline] [Order article via Infotrieve] |
25. | Reinton, N., Collas, P., Haugen, T. B., Skalhegg, B. S., Hansson, V., Jahnsen, T., and Tasken, K. (2000) Dev. Biol. 223, 194-204[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Vijayaraghavan, S.,
Goueli, S. A.,
Davey, M. P.,
and Carr, D. W.
(1997)
J. Biol. Chem.
272,
4747-4752 |
27. |
Vijayaraghavan, S.,
Liberty, G. A.,
Mohan, J.,
Winfrey, V. P.,
Olson, G. E.,
and Carr, D. W.
(1999)
Mol. Endocrinol.
13,
705-717 |
28. |
Burton, K. A.,
Treash-Osio, B.,
Muller, C. H.,
Dunphy, E. L.,
and McKnight, G. S.
(1999)
J. Biol. Chem.
274,
24131-24136 |
29. | Ling, M., Merante, F., and Robinson, B. H. (1995) Nucleic Acids Res. 23, 4924-4925[Medline] [Order article via Infotrieve] |
30. | Sathe, G. M., O'Brien, S., McLaughlin, M. M., Watson, F., and Livi, G. P. (1991) Nucleic Acids Res. 19, 4775[Medline] [Order article via Infotrieve] |
31. | Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Fujita, A.,
Nakamura, K.,
Kato, T.,
Watanabe, N.,
Ishizaki, T.,
Kimura, K.,
Mizoguchi, A.,
and Narumiya, S.
(2000)
J. Cell Sci.
113,
103-112 |
33. | Kong, M., Richardson, R. T., Widgren, E. E., and O'Rand, M. G. (1995) Biol. Reprod. 53, 579-590[Abstract] |
34. | Lea, I. A., Richardson, R. T., Widgren, E. E., and O'Rand, M. G. (1996) Biochim. Biophys. Acta 1307, 263-266[Medline] [Order article via Infotrieve] |
35. |
Hausken, Z. E.,
Dell'Acqua, M. L.,
Coghlan, V. M.,
and Scott, J. D.
(1996)
J. Biol. Chem.
271,
29016-29022 |
36. |
Li, Y.,
and Rubin, C. S.
(1995)
J. Biol. Chem.
270,
1935-1944 |
37. | Newlon, M. G., Roy, M., Morikis, D., Hausken, Z. E., Coghlan, V., Scott, J. D., and Jennings, P. A. (1999) Nat. Struct. Biol. 6, 222-227[CrossRef][Medline] [Order article via Infotrieve] |
38. | Kaibuchi, K., Kuroda, S., and Amano, M. (1999) Annu. Rev. Biochem. 68, 459-486[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Somlyo, A. P.,
and Somlyo, A. V.
(2000)
J. Physiol. (Lond)
522,
177-185 |
40. | Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., Yamamori, B., Feng, J., Nakano, T., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 273, 245-248[Abstract] |
41. | Hinsch, K. D., Habermann, B., Just, I., Hinsch, E., Pfisterer, S., Schill, W. B., and Aktories, K. (1993) FEBS Lett. 334, 32-36[CrossRef][Medline] [Order article via Infotrieve] |
42. | Smith, G. D., Wolf, D. P., Trautman, K. C., da Cruz e Silva, E. F., Greengard, P., and Vijayaraghavan, S. (1996) Biol. Reprod. 54, 719-727[Abstract] |
43. | Vijayaraghavan, S., Stephens, D. T., Trautman, K., Smith, G. D., Khatra, B., da Cruz e Silva, E. F., and Greengard, P. (1996) Biol. Reprod. 54, 709-718[Abstract] |
44. |
Schillace, R. V.,
and Scott, J. D.
(1999)
J. Clin. Invest.
103,
761-765 |