BACKGROUND
cyclic 3', 5'-adenosine monophosphate  is a key intracellular signaling molecule, whose main function is to activate the camp-dependent protein kinase   <cit> .
pka is a heterotetrameric holoenzyme consisting of a regulatory  subunit dimer and two catalytic  subunits.
the holoenzyme is activated when four molecules of camp bind to the r subunit dimer, two to each r subunit, releasing two free active c subunits .
in man, four different r subunits designated riα, riβ riiα, riiβ , and four different c subunits  have been identified  <cit> .
the cα and cβ subunits are expressed in most tissues, while the cγ subunit, which is transcribed from an intron-less gene, represents a retroposon derived from the cα subunit  <cit> .
cγ is only expressed in human testis  <cit> .
prkx is an x chromosome-encoded protein kinase, and was identified as a pka c subunit since it is inhibited by both pki and riα, and the riα/prkx complex is activated by camp  <cit> .
splice variants of both cα and cβ have been identified.
in the case of cα, two splice variants have been identified and termed cα <dig>  <cit>  and cα <dig>  <cit> .
cα <dig> and cα <dig> have non-identical n-terminal ends encoded by alternative use of two exons  located upstream of exon  <dig> in the murine cα gene.
the cβ gene encodes a number of products identified in various species and have been designated cβ <dig>  cβ <dig>  cβ <dig>  cβ <dig>  cβ3ab, cβ3b, cβ3abc, cβ4ab, cβ4b, cβ4abc  <cit> .
as is the case for the cα forms, all the cβ variants have variable n-terminal ends which are encoded by different exons upstream of exon  <dig> in the cβ gene .
pka-c splice variants are tissue-specifically expressed and some experimental evidence support that they may harbor specific features and non-identical activities when associated with the r subunits to form holoenzymes .
with regard to this cα <dig> it is the sole c subunit expressed in the sperm cell.
moreover, cα <dig> was shown to be vital for mouse sperm motility since ablation rendered the sperm cells non-motile and the male individuals infertile .
cα <dig> is equipped with an n-terminal of  <dig> amino acids which undergo three well defined co- and posttranslational modifications.
they include in vivo myristylation of gly <dig>  <cit> .
at position + <dig> an asn is encoded which is partly deamidated in vivo leading to cα1-asp <dig> and cα1-isoasp <dig>  <cit> .
a third modification is identified as a pka-autophosphorylation site at ser <dig>  <cit> .
cα <dig> on the other hand is encoded with  <dig> unique amino acids at the n-terminus which to our knowledge do not have the ability to undergo any of the n-terminal modifications seen for cα <dig>
based on the different n-terminal sequences of cα <dig> and cα <dig> we speculate that they will introduce distinct biological features to these subunits.
to investigate this hypothesis we made a thorough characterization of cα <dig> activities both in vivo and in vitro and compared the results to what is known for cα <dig> and to results obtained for cα <dig> in the present work.
methods
sperm cell isolation
semen samples were obtained from patients attending infertility investigations at the andrology laboratory at rikshospitalet-radiumhospitalet hf, oslo, norway.
all patients signed a letter of approval and all experiments were done according to the recommendation from the regional committees for medical and health research ethics.
all men produced their ejaculates on site or at home after 3- <dig> days of sexual abstinence.
samples were collected by masturbation into a wide-mouthed sterile container  and after  <dig> min of liquefaction at 37°c, sperm parameters were evaluated according to world health organization  methods , cambridge university press, cambridge .
sperm cells were isolated from the seminal plasma by percoll gradient centrifugation.
sperm samples were pipetted on top of a 90%/45% percoll gradient and centrifuged at  <dig> rpm for  <dig> min, no brake.
after centrifugation, the sperm pellet was recovered by first using a sterile glass pasteur pipette to remove the top layers of the semen sample and sperm gradient, leaving approximately  <dig>  ml of the bottom layer.
the sperm pellet was subsequently resuspended and washed twice in phosphate buffered saline  and centrifuged again at  <dig> rpm for  <dig> min.
sperm head and tail separation
isolated sperm cells were diluted to  <dig> mill/ml in pbs and sonicated mildly for  <dig> sec at low frequency.
ten μl samples were taken out to be examined by microscopy to assure head and tail separation.
after complete separation the mixture was centrifuged at  <dig> g for  <dig> min.
the supernatant containing the tails was transferred to a new tube and tails pelleted by centrifugation at  <dig>  × g for  <dig> min.
the tail pellet and the pellet from the first centrifugation were separately solubelized in ripa buffer  containing  <dig> mm dtt,  <dig> mm pmsf and protease inhibitor cocktail , and sonicated  <dig> ×  <dig> seconds at full effect.
sds-polyacrylamide gel electrophoresis
sds-page, was performed as described by  <cit> .
briefly, samples were diluted in sds sample buffer , boiled for  <dig> min and loaded onto slab gels consisting of a  <dig> % stacking gel and a  <dig> % separating gel.
immunoprecipitation
lysates were cleared by centrifugation at  <dig> g for  <dig> min at 4°c, and subsequently incubated with primary antibody  for  <dig> h to overnight.
antibody-antigen complexes were precipitated using either dynabeads protein g , anti-mouse agarose beads or anti-rabbit agarose beads .
precipitates were washed three times using appropriate buffer and extracted with buffer in the presence or absence of  <dig> mm 8-cpt camp as indicated in the figure legends.
immunoblot analysis
total protein was estimated by bradford protein assay .
proteins were separated by sds-page and transferred to pvdf membranes by electro blotting.
membranes were blocked in 5% skimmed milk powder in tris-buffered saline containing  <dig> % tween- <dig>  for  <dig> h at room temperature, and then incubated for  <dig> h at room temperature or overnight at 4°c with the appropriate primary antibodies diluted in tbst rabbit polyclonal anti-c 1: <dig> , anti-cα <dig> , mouse anti-riα 1: <dig> and rabbit anti-riiα serum  <cit> .
membranes were washed for about  <dig> h in tbst and further incubated with hrp-conjugated secondary antibodies .
membranes were washed and finally developed using supersignal© west pico chemiluminescent .
endogenous sperm cell protein fractionation
sperm cells were homogenized in pe buffer  containing  <dig> % triton x- <dig>   <dig> mm dtt,  <dig> mm pmsf and protease inhibitor cocktail , and sonicated  <dig> ×  <dig> seconds.
deae cellulose  was applied to a column and equilibrated with pe buffer with  <dig> mm sucrose.
the sperm cell homogenates were cleared by centrifugation at  <dig>  g for  <dig> min at 4°c and applied to the column.
after washing with pe buffer, the column was eluted with a linear 0- <dig> mm nacl gradient created by a gradient mixer ,  <dig> ml fractions were collected and salt concentration checked by refractometry.
every other fraction, starting with fraction  <dig> was subjected to phosphotransferase activity measurements in the absence and presence of camp.
peak fractions were concentrated to  <dig> μl using centrifugal filters with  <dig> kda cutoff  and subjected to immunoblot analysis.
phosphotransferase assay
catalytic activity of pka was assayed by phosphorylating the pka specific substrate kemptide  using γ-atp  as previously described  <cit> .
specific activity is determined as u/mg, which is defined as μmol/mg × min.
camp-binding assay
determination of specific camp-binding of soluble r subunits was carried out in a buffer containing  <dig>  μmol -camp   <cit> .
activation assay
the activation assay of cα <dig> and myrcα <dig> with riα and riiβ, respectively, was performed in a spectrophotometric kinase activity assay as described earlier  <cit> .
expression of cα <dig>  cα <dig> and myristylated cα1
recombinant non-myristylated human cα <dig> was expressed and purified as described previously .
recombinant human cα <dig>  as well as recombinant human ca <dig>  were co-transformed with n-myristyl-transferase in escherichia coli bl <dig>   and co-expressed with human cα <dig> as well as cα <dig> using the same conditions.
both proteins were purified by affinity chromatography using pki-peptide affi-gel.
the procedure was first described by olsen et al.
<cit>  and modified after thullner et al.
<cit> .
expression and purification of r subunits
recombinant human r subunits  were over-expressed in escherichia coli bl <dig>  ril  and purified according to a procedure by bertinetti et al.
<cit>  using sp-8-aea-camps-agarose.
the purity of the r and c proteins was confirmed by sds-page as well as by immunoblot analysis and the biological activity of the proteins was measured as described before  <cit> .
primary sequence of cα <dig> and the presence of n-myristylation at cα <dig> were checked by mass spectrometry .
spr analysis
all spr interaction analyses were performed at 25°c in  <dig> mm mops ph  <dig>   <dig> mm nacl plus  <dig> %  surfactant p <dig>   <dig> mm atp,  <dig> mm mgcl <dig> and  <dig> μm edta using biacore  <dig> or  <dig> instruments .
for covalent coupling of the c subunits, carboxymethylated sensor chip surfaces  were activated with nhs/edc for  <dig> min and non-myristylated cα <dig>  cα <dig> and myrcα <dig>  as described  <cit>  were injected on separate flow cells with a flow rate of  <dig> μl/min until approximately  <dig> response units   <cit>  were reached.
this amine coupling was described previously .
deactivation of the surface was performed using  <dig> m ethanolamine-hcl  for  <dig> min.
as a reference one flow cell was activated and deactivated in the absence of any protein.
the interaction experiments with four r subunit were performed at 25°c in running buffer  at a flow rate of  <dig> μl/min.
during simultaneous r subunit injection over the four surfaces , the dissociation phase was monitored for  <dig> sec.
the binding of r subunit to c subunit was not limited by mass transport according to previous experiments  <cit> .
response of the activated/deactivated reference cell was subtracted.
surfaces were regenerated with two subsequent  <dig> min injections of  <dig>  mm camp,  <dig>  mm edta in running buffer.
evaluation of non-normalized data was performed with biaevaluation  <dig>  rc <dig> .
a langmuir 1: <dig> binding model was applied for the kinetic analysis of c subunit/r subunit interactions  <cit> .
the sensor chip was stored at 4°c in running buffer and tested for proper performance prior to each analysis.
RESULTS
sperm cell-specific cα <dig> associates with riα and riiα in a camp-dependent fashion to form pkai and pkaii holoenzymes in vivo
we first determined whether human sperm cells express cα <dig>  protein extracts of t cells, whole sperm, sperm tail, and sperm heads were separated by sds-page transferred to immunoblot filters and filters probed with a pan-c antibody  and an anti-cα <dig> antiserum .
this confirms previous results that cα <dig> is not expressed in t cells and is distributed in tail and head of spermatozoa  <cit> .
ri and rii are both expressed in all sperm cell compartments and are known to form pka type i and type ii   <cit> .
pkai and pkaii holoenzymes can be separated by deae ion exchange chromatography using increasing concentrations of nacl  <cit> .
since cα <dig> is the sole c subunit in human sperm cells it is expected that it will associate with ri and rii to form pkai and pkaii, respectively, as has been demonstrated in the mouse  <cit> .
to test this, whole sperm cell extracts  were fractionated on deae resins by a linear nacl gradient ranging from  <dig> to  <dig> mm).
two peaks of phosphotransferase-  and camp-binding activity  between 50- <dig> mm and 100- <dig> mm nacl, respectively, were observed.
this implied formation of both pkai and pkaii .
c and r subunit identity were documented by immunoblotting using a pan-c antibody  and anti-cα <dig>  as well as anti-riα  and anti-riiα .
this showed that immunoreactive cα <dig> co-elutes with the two major r subunits in sperm cells, riα  and riiα   <cit> .
from the figure it is also seen that some of the c activity was detected before the r subunit activity in the first peak, implying free c subunit.
moreover, we also noted that pkai and pkaii containing cα <dig> eluted at comparable concentrations of nacl.
to further investigate whether human cα <dig> forms pkai and pkaii in cell extracts we immunoprecipitated  with anti-riα and anti-riiα .
using immunoblotting and anti-cα <dig>  and anti- pan-c  we showed that both riα and riiα associate with cα <dig>  implying that cα <dig> forms pkai and ii in vivo .
to define whether the r-c interaction is specific and functional, the iped proteins were challenged with camp to dissociate the holoenzyme into r subunit dimers and free c subunits  <cit> .
in these experiments the iped r subunits would be expected to be immobilized by the precipitating antiserum and remain in the pellet  after the camp wash whereas the iped c subunit would be released into the supernatant  in the presence of camp .
figure  <dig>  depicts that immunoreactive cα <dig> is released into the supernatant after the camp wash  after both anti-riα and anti-riiα ip  whereas the immunoreactive cα <dig> subunit remained in the pellet  in the absence of camp  .
comparable activities of cα <dig> and cα1
our results and several reports showing that cα <dig> associates with both ri and rii subunits  in a camp-sensitive fashion imply that n-terminal differences do not interfere with pka holoenzyme formation.. to further investigate wethercα <dig> has activities that differ from cα <dig> we expressed cα <dig> using the prest b vector.
first we noted that cα <dig> and cα <dig> were captured in the soluble and particulate fractions of the bacteria lysates, respectively, suggesting differences in solubility .
furthermore, specific activity of expressed cα <dig> was determined to  <dig> ±  <dig> units/mg  which was notably lower than the specific activity of expressed cα <dig> .
taken together this may imply differential features of cα <dig> and cα <dig>  based on this and to determine the exact activities for cα <dig> three features were investigated.
these included  cα2's substrate affinity,  the ability to form holoenzymes with ri and rii in vitro and  the km values for kemptide and atp.
in the latter case we found the km values of cα <dig> for kemptide and atp to be  <dig>   <dig> ±  <dig>  μm and  <dig>  ±  <dig>  μm, respectively .
this is in good agreement with previous results obtained with expressed cα <dig>  <cit> .
we next monitored camp-sensitivity of type i and ii pka holoenzymes containing cα <dig> in vitro.
to calculate the accurate camp activation constant, pkai  and pkaii  containing cα <dig> holoenzymes were incubated with  <dig> μm kemptide and  <dig> mm atp in the presence of increasing concentrations of camp.
kact values for camp were  <dig> nm and  <dig> nm for the holoenzyme formed with riα and riiβ, respectively, showing that pkai containing cα <dig> are nearly  <dig> fold more sensitive to camp than pkaii containing cα <dig> holoenzymes .
the corresponding values for holoenzymes formed with myrcα <dig> were  <dig> nm  and  <dig> nm   again demonstrating a  <dig> fold increased sensitivity for the ri holoenzyme.
this is also in agreement with previous in vitro results for pkai and ii holoenzymes containing cα <dig> , and the kact values for mouse pkai isolated from sperm cells ablated for pkaii   <cit> .
it should be noted that kact in wild type sperm cells which mainly express pkaii   <cit>  is almost identical to the kact in riiα ablated sperm cells.
this may suggest that pkai and pkaii display comparable kact's in vivo and hence contradicts the in vitro results demonstrated previously  <cit>  and by us here.
it should also be noted that pkai although expressed at low levels may skew the observed kact values, due to its sensitivity for camp.
to what extent this has biological consequences as has been demonstrated for pkai and pkaii in lymphoid cells , remains to be tested.
we then investigated the potency of the r subunits  and the protein kinase inhibitor pkiα to inhibit cα <dig> phosphotransferase activity in vitro.
purified cα <dig>  was mixed with a fixed concentration of kemptide  in the presence of increasing concentrations of the various r subunits or pkiα.
all the r subunits inhibited cα2-dependent kinase activity by 50% at  <dig> nm and showed complete inhibition at a 1: <dig> molar ratio .
a fixed dose  of cα <dig> was inhibited by pkiα in a dose-dependent manner with complete inhibition at stoichiometric concentrations of cα <dig> and pkiα .
the inhibitory effects of the various r subunits and pki have previously been determined for cα <dig>  and indicate that the efficiency in inhibiting cα <dig> and cα <dig> is similar for all r subunits and pkiα.
using a biacore technology we next investigated the dissociation equilibrium constants , association  and dissociation  rate constants for the various r subunits in association with either cα <dig> or cα <dig>  we immobilized  <dig> rus of myristylated cα <dig>  and cα <dig> on separate flow cells of a cm <dig> biacore sensor chip.
unmyristylated cα <dig> was used as reference .
in the presence of  <dig> mm atp and  <dig> mm mgcl <dig> the r subunits were simultaneously run over both c subunits on the sensor chip at a flow rate of  <dig> μl/min.
in the case of riα and riβ they were run over the sensor chip at concentrations between  <dig>  and  <dig> nm, and riiα and riiβ between  <dig>  to  <dig> nm .
figure  <dig>  shows representative runs of riα, riβ, riiα and riiβ  on cα <dig> and myrcα <dig>  the shape of the curves indicates that the relative on and off rates for riα when associated with either cα <dig> or myrcα <dig> were highly similar.
the relative kass values were slightly different,  <dig>  ×  <dig> and  <dig>  ×  <dig> m-1s- <dig> for riα versus myrcα <dig> and cα <dig>  respectively.
the same was true for riβ.
however, in this case, although the kd value was almost identical to riα, the association as well as the dissociation rate constants for riβ was  <dig> times faster.
finally, no differences could be observed for the interaction of riiα and riiβ against myrcα <dig> versus cα <dig> .
in order to investigate the binding behavior of pki, gst-pkiα was immobilized on sensor chips as described previously  <cit> , and various concentrations of cα <dig>  cα <dig>  myrcα <dig> and, for comparison, mouse cα <dig> were run at a flow rate of  <dig> μl/min over the sensor chips.
this revealed a kd for all c subunits and pkiα at a range around  <dig>  nm where the myrcα <dig> displayed a slightly faster association rate compared to cα <dig>  with all the dissociation rates being similar  .
spr measurements with single concentrations demonstrated almost identical shapes of the curves , indicating comparable association and dissociation rates.
in order to determine accurate association rate constants, different concentrations of the respective c subunits were applied , leading again to the conclusion that the binding activities of cα <dig> and cα <dig> for pkiα are highly similar.
discussion
at the protein level cα <dig> and cα <dig> are 97% homologous and only differ at the n-terminal end.
based on this we investigated to what extent differences at the n-terminus may influence splice variant-specific activities that may have biological importance.
we found that cα <dig> expressed in bacteria was not captured by inclusion bodies as was the case with cα <dig>  moreover, the specific activity of cα <dig> was lower compared to cα <dig>  apart from these differences we observed that cα <dig> was highly similar to cα <dig> in all parameters measured.
this included association of cα <dig> with ri and rii to form camp-sensitive holoenzymes both in vivo and in vitro.
furthermore, km values of cα <dig> for kemptide and atp were comparable to those determined for cα <dig>  this was also the case for the ability of ri, rii and pki to inhibit cα <dig> enzyme activity in vitro .
finally, kd values as measured by spr were shown to be comparable between cα <dig> and cα <dig> towards the ri and rii subunits as well as pki.
several reports imply that n-terminal modifications of cα <dig> introduce specific features that may have biological consequences.
to this end it has been suggested that phosphorylation of ser <dig> in cα <dig> introduces an electrostatic force which may help the c subunit to remain soluble even when myristylated .
moreover, two reports have demonstrated that the n-terminal myristyl moiety of cα <dig> is embedded in a hydrophobic pocket encompassed in the large lobe .
mutation of gly <dig> to ala rendering the cα <dig> non-myristylated, demonstrated that myristylation was non-essential for conformation and enzyme activation, and was not required for cα <dig> interaction with other proteins including various substrates and the r subunits  <cit> .
the fact that cα <dig> is not myristylated and displays comparable activities with myristylated cα <dig> suggests that myristylation is not essential for catalytic activity, holoenzyme formation and inhibition by pki.
this is further supported in that deletion of the entire cα <dig> n-terminus did not severely interfere with catalytic activity and inhibitor binding despite that deletion caused thermo instability  <cit> .
this also suggests that the amino acids  <dig>  and  <dig>  of cα <dig> are not essential for activity a suggestion which is supported by our results on cα <dig>
cα <dig> and cβ <dig> which are 100% identical at the n-terminus, but only 91% identical in the sequence encode by exon  <dig> through  <dig>  have different apparent sizes  and possess distinct biochemical properties both in vitro and in vivo .
these differences include differential km values for certain peptide substrates and that cα <dig> but not cβ <dig> is inhibited by substrate concentrations above  <dig> μm.
in addition, they display distinct ic <dig> values for pki and riiα.
taken together with our results this may suggest that the amino acid sequence encoded by exon  <dig> through  <dig> and not exon  <dig> influence c subunit activities such as holoenzyme formation, enzyme activity and inhibition by r and pki.
this may further imply that cα <dig> and cβ <dig> have distinct roles in regulating cellular processes.
this was recently shown in t cells which express cα <dig>  cβ <dig> and cβ <dig>  in that study cα <dig>  but none of the cβ forms, mediated the inhibitory effect of camp on immune cell reactivity in vivo .
in light of these observations it is also of interest to note that cα <dig> but not cα <dig> is required for sperm cell forward velocity and male fertility, despite 100% identity at the amino acid sequence encoded by exon  <dig> through  <dig> .
however, since cα <dig> is the sole c subunit in sperm cells , the difference observed may only be ascribed to tissue-specific expression and not sequence-specific differences.
in contrast to cα <dig> it is expected that the hydrophobic pocket in which the myristyl group is submersed in cα <dig>  is constitutively empty and exposed to the surroundings at all time in non-myristylated cα <dig>  it has been speculated whether exposure of the hydrophobic pocket would introduce more lipophilic properties to the cα <dig> subunit  <cit> .
support for such a hypothesis is found in a by a study demonstrating that binding of cα <dig> to rii induced a unique conformation that is associated with exposure of the hydrophobic pocket to the surroundings due to increase in n-terminal flexibility of the n-myristate away from the large lobe.
this renders cα <dig> more hydrophobic and promotes membrane association of the pka ii holoenzyme only  <cit> .
therefore it may be suggested that exposure of the hydrophobic pocket serves features such as isoform specific features and subcellular localization of the c subunit.
to this end it is interesting to note that cα <dig> is associated with the sperm tail in the presence of detergent treatment with 1% triton x- <dig> .and, after a challenge with  <dig> mm camp  <cit> .
this may be indicative of a direct association of cα <dig> with subcellular structures.
to what extent such attachment involves the hydrophobic pocket remains unknown.
in other cells and tissues, c subunits targeted to subcellular structures independent of the r subunit and traditional a-kinase anchoring proteins have been demonstrated.
to day a number of c subunit binding proteins have been identified.
these include pki, a-kinase interacting protein  <dig> , homologous to akap <dig> , inhibitor of nfkappab kinase , caveolin- <dig> and p <dig> neutrophine receptor   <cit> .
to what extent cα <dig> is targeted to the sperm cell midpiece through a c interaction protein and if specificity of binding is retained in the hyper variable n-terminal end remains to be shown.
however, it should be noted that deamination of the asn <dig> moiety in cα <dig> helps fine-tuning enzyme distribution within the cell in vivo  <cit> .
moreover, p75ntr was shown to specifically bind to the cβ splice variant cβ4ab  <cit> , which is encoded with unique n-terminal domain that may not undergo the same posttranslational modifications as cα <dig> .
together this may imply that the n-terminal end may be important for targeting and specificity of subcellular localization of the various c subunits.
CONCLUSIONS
our study demonstrates that n-terminal sequence encoded by alternative use of exons upstream of exon  <dig> in the prkaca gene does not influence c subunit activities such as holoenzyme formation, camp sensitivity, enzyme activity as well as inhibition by ri, rii and pki.
based on several studies it may be suggested that the n-terminus is involved in other c subunit features such as subcellular localization.
authors' contributions
mmv has written the manuscript and has designed an contributed to the experiments in figures  <dig> through  <dig> and table  <dig> and  <dig>  hmz has contributed to the biacore experiments.
em has performed the experiments in figures  <dig> and  <dig>  hvb has made recombinant proteins for cα <dig> and cα <dig> and contributed to the experiments in figure  <dig>  fwh and bss have conceived the ideas to the manuscript and contributed to the writing.
all authors read and approved the final manuscript.
abbreviations
pka: protein kinase a; c: catalytic subunit; cα: alpha-form of c; cα2: sperm-specific c subunit.

 cyclic 3', 5'-adenosine monophosphate is a key intracellular signaling molecule, whose main function is to activate the camp-dependent protein kinase. pka is a heterotetrameric holoenzyme consisting of a regulatory subunit dimer and two catalytic subunits. the holoenzyme is activated when four molecules of camp bind to the r subunit dimer, two to each r subunit, releasing two free active c subunits. in man, four different r subunits designated riα, riβ riiα, riiβ, and four different c subunits have been identified. the cα and cβ subunits are expressed in most tissues, while the cγ subunit, which is transcribed from an intron-less gene, represents a retroposon derived from the cα subunit. cγ is only expressed in human testis. prkx is an x chromosome-encoded protein kinase, and was identified as a pka c subunit since it is inhibited by both pki and riα, and the riα/prkx complex is activated by camp. splice variants of both cα and cβ have been identified. in the case of cα, two splice variants have been identified and termed cα and cα. cα and cα have non-identical n-terminal ends encoded by alternative use of two exons located upstream of exon in the murine cα gene. the cβ gene encodes a number of products identified in various species and have been designated cβ cβ cβ cβ cβ3ab, cβ3b, cβ3abc, cβ4ab, cβ4b, cβ4abc. as is the case for the cα forms, all the cβ variants have variable n-terminal ends which are encoded by different exons upstream of exon in the cβ gene. pka-c splice variants are tissue-specifically expressed and some experimental evidence support that they may harbor specific features and non-identical activities when associated with the r subunits to form holoenzymes. with regard to this cα it is the sole c subunit expressed in the sperm cell. moreover, cα was shown to be vital for mouse sperm motility since ablation rendered the sperm cells non-motile and the male individuals infertile. cα is equipped with an n-terminal of amino acids which undergo three well defined co- and posttranslational modifications. they include in vivo myristylation of gly. at position + an asn is encoded which is partly deamidated in vivo leading to cα1-asp and cα1-isoasp. a third modification is identified as a pka-autophosphorylation site at ser. cα on the other hand is encoded with unique amino acids at the n-terminus which to our knowledge do not have the ability to undergo any of the n-terminal modifications seen for cα based on the different n-terminal sequences of cα and cα we speculate that they will introduce distinct biological features to these subunits. to investigate this hypothesis we made a thorough characterization of cα activities both in vivo and in vitro and compared the results to what is known for cα and to results obtained for cα in the present work. methods sperm cell isolation semen samples were obtained from patients attending infertility investigations at the andrology laboratory at rikshospitalet-radiumhospitalet hf, oslo, norway. all patients signed a letter of approval and all experiments were done according to the recommendation from the regional committees for medical and health research ethics. all men produced their ejaculates on site or at home after 3- days of sexual abstinence. samples were collected by masturbation into a wide-mouthed sterile container and after min of liquefaction at 37°c, sperm parameters were evaluated according to world health organization methods, cambridge university press, cambridge. sperm cells were isolated from the seminal plasma by percoll gradient centrifugation. sperm samples were pipetted on top of a 90%/45% percoll gradient and centrifuged at rpm for min, no brake. after centrifugation, the sperm pellet was recovered by first using a sterile glass pasteur pipette to remove the top layers of the semen sample and sperm gradient, leaving approximately ml of the bottom layer. the sperm pellet was subsequently resuspended and washed twice in phosphate buffered saline and centrifuged again at rpm for min. sperm head and tail separation isolated sperm cells were diluted to mill/ml in pbs and sonicated mildly for sec at low frequency. ten μl samples were taken out to be examined by microscopy to assure head and tail separation. after complete separation the mixture was centrifuged at g for min. the supernatant containing the tails was transferred to a new tube and tails pelleted by centrifugation at × g for min. the tail pellet and the pellet from the first centrifugation were separately solubelized in ripa buffer containing mm dtt, mm pmsf and protease inhibitor cocktail, and sonicated × seconds at full effect. sds-polyacrylamide gel electrophoresis sds-page, was performed as described by. briefly, samples were diluted in sds sample buffer, boiled for min and loaded onto slab gels consisting of a % stacking gel and a % separating gel. immunoprecipitation lysates were cleared by centrifugation at g for min at 4°c, and subsequently incubated with primary antibody for h to overnight. antibody-antigen complexes were precipitated using either dynabeads protein g, anti-mouse agarose beads or anti-rabbit agarose beads. precipitates were washed three times using appropriate buffer and extracted with buffer in the presence or absence of mm 8-cpt camp as indicated in the figure legends. immunoblot analysis total protein was estimated by bradford protein assay. proteins were separated by sds-page and transferred to pvdf membranes by electro blotting. membranes were blocked in 5% skimmed milk powder in tris-buffered saline containing % tween- for h at room temperature, and then incubated for h at room temperature or overnight at 4°c with the appropriate primary antibodies diluted in tbst rabbit polyclonal anti-c 1:, anti-cα, mouse anti-riα 1: and rabbit anti-riiα serum. membranes were washed for about h in tbst and further incubated with hrp-conjugated secondary antibodies. membranes were washed and finally developed using supersignal© west pico chemiluminescent. endogenous sperm cell protein fractionation sperm cells were homogenized in pe buffer containing % triton x- mm dtt, mm pmsf and protease inhibitor cocktail, and sonicated × seconds. deae cellulose was applied to a column and equilibrated with pe buffer with mm sucrose. the sperm cell homogenates were cleared by centrifugation at g for min at 4°c and applied to the column. after washing with pe buffer, the column was eluted with a linear 0- mm nacl gradient created by a gradient mixer, ml fractions were collected and salt concentration checked by refractometry. every other fraction, starting with fraction was subjected to phosphotransferase activity measurements in the absence and presence of camp. peak fractions were concentrated to μl using centrifugal filters with kda cutoff and subjected to immunoblot analysis. phosphotransferase assay catalytic activity of pka was assayed by phosphorylating the pka specific substrate kemptide using γ-atp as previously described. specific activity is determined as u/mg, which is defined as μmol/mg × min. camp-binding assay determination of specific camp-binding of soluble r subunits was carried out in a buffer containing μmol -camp. activation assay the activation assay of cα and myrcα with riα and riiβ, respectively, was performed in a spectrophotometric kinase activity assay as described earlier. expression of cα cα and myristylated cα1 recombinant non-myristylated human cα was expressed and purified as described previously. recombinant human cα as well as recombinant human ca were co-transformed with n-myristyl-transferase in escherichia coli bl and co-expressed with human cα as well as cα using the same conditions. both proteins were purified by affinity chromatography using pki-peptide affi-gel. the procedure was first described by olsen et al. and modified after thullner et al.. expression and purification of r subunits recombinant human r subunits were over-expressed in escherichia coli bl ril and purified according to a procedure by bertinetti et al. using sp-8-aea-camps-agarose. the purity of the r and c proteins was confirmed by sds-page as well as by immunoblot analysis and the biological activity of the proteins was measured as described before. primary sequence of cα and the presence of n-myristylation at cα were checked by mass spectrometry. spr analysis all spr interaction analyses were performed at 25°c in mm mops ph mm nacl plus % surfactant p mm atp, mm mgcl and μm edta using biacore or instruments. for covalent coupling of the c subunits, carboxymethylated sensor chip surfaces were activated with nhs/edc for min and non-myristylated cα cα and myrcα as described were injected on separate flow cells with a flow rate of μl/min until approximately response units were reached. this amine coupling was described previously. deactivation of the surface was performed using m ethanolamine-hcl for min. as a reference one flow cell was activated and deactivated in the absence of any protein. the interaction experiments with four r subunit were performed at 25°c in running buffer at a flow rate of μl/min. during simultaneous r subunit injection over the four surfaces, the dissociation phase was monitored for sec. the binding of r subunit to c subunit was not limited by mass transport according to previous experiments. response of the activated/deactivated reference cell was subtracted. surfaces were regenerated with two subsequent min injections of mm camp, mm edta in running buffer. evaluation of non-normalized data was performed with biaevaluation rc. a langmuir 1: binding model was applied for the kinetic analysis of c subunit/r subunit interactions. the sensor chip was stored at 4°c in running buffer and tested for proper performance prior to each analysis.  sperm cell-specific cα associates with riα and riiα in a camp-dependent fashion to form pkai and pkaii holoenzymes in vivo we first determined whether human sperm cells express cα protein extracts of t cells, whole sperm, sperm tail, and sperm heads were separated by sds-page transferred to immunoblot filters and filters probed with a pan-c antibody and an anti-cα antiserum. this confirms previous results that cα is not expressed in t cells and is distributed in tail and head of spermatozoa. ri and rii are both expressed in all sperm cell compartments and are known to form pka type i and type ii. pkai and pkaii holoenzymes can be separated by deae ion exchange chromatography using increasing concentrations of nacl. since cα is the sole c subunit in human sperm cells it is expected that it will associate with ri and rii to form pkai and pkaii, respectively, as has been demonstrated in the mouse. to test this, whole sperm cell extracts were fractionated on deae resins by a linear nacl gradient ranging from to mm). two peaks of phosphotransferase- and camp-binding activity between 50- mm and 100- mm nacl, respectively, were observed. this implied formation of both pkai and pkaii. c and r subunit identity were documented by immunoblotting using a pan-c antibody and anti-cα as well as anti-riα and anti-riiα. this showed that immunoreactive cα co-elutes with the two major r subunits in sperm cells, riα and riiα. from the figure it is also seen that some of the c activity was detected before the r subunit activity in the first peak, implying free c subunit. moreover, we also noted that pkai and pkaii containing cα eluted at comparable concentrations of nacl. to further investigate whether human cα forms pkai and pkaii in cell extracts we immunoprecipitated with anti-riα and anti-riiα. using immunoblotting and anti-cα and anti- pan-c we showed that both riα and riiα associate with cα implying that cα forms pkai and ii in vivo. to define whether the r-c interaction is specific and functional, the iped proteins were challenged with camp to dissociate the holoenzyme into r subunit dimers and free c subunits. in these experiments the iped r subunits would be expected to be immobilized by the precipitating antiserum and remain in the pellet after the camp wash whereas the iped c subunit would be released into the supernatant in the presence of camp. figure depicts that immunoreactive cα is released into the supernatant after the camp wash after both anti-riα and anti-riiα ip whereas the immunoreactive cα subunit remained in the pellet in the absence of camp. comparable activities of cα and cα1 our results and several reports showing that cα associates with both ri and rii subunits in a camp-sensitive fashion imply that n-terminal differences do not interfere with pka holoenzyme formation.. to further investigate wethercα has activities that differ from cα we expressed cα using the prest b vector. first we noted that cα and cα were captured in the soluble and particulate fractions of the bacteria lysates, respectively, suggesting differences in solubility. furthermore, specific activity of expressed cα was determined to ± units/mg which was notably lower than the specific activity of expressed cα. taken together this may imply differential features of cα and cα based on this and to determine the exact activities for cα three features were investigated. these included cα2's substrate affinity, the ability to form holoenzymes with ri and rii in vitro and the km values for kemptide and atp. in the latter case we found the km values of cα for kemptide and atp to be ± μm and ± μm, respectively. this is in good agreement with previous results obtained with expressed cα. we next monitored camp-sensitivity of type i and ii pka holoenzymes containing cα in vitro. to calculate the accurate camp activation constant, pkai and pkaii containing cα holoenzymes were incubated with μm kemptide and mm atp in the presence of increasing concentrations of camp. kact values for camp were nm and nm for the holoenzyme formed with riα and riiβ, respectively, showing that pkai containing cα are nearly fold more sensitive to camp than pkaii containing cα holoenzymes. the corresponding values for holoenzymes formed with myrcα were nm and nm again demonstrating a fold increased sensitivity for the ri holoenzyme. this is also in agreement with previous in vitro results for pkai and ii holoenzymes containing cα, and the kact values for mouse pkai isolated from sperm cells ablated for pkaii. it should be noted that kact in wild type sperm cells which mainly express pkaii is almost identical to the kact in riiα ablated sperm cells. this may suggest that pkai and pkaii display comparable kact's in vivo and hence contradicts the in vitro results demonstrated previously and by us here. it should also be noted that pkai although expressed at low levels may skew the observed kact values, due to its sensitivity for camp. to what extent this has biological consequences as has been demonstrated for pkai and pkaii in lymphoid cells, remains to be tested. we then investigated the potency of the r subunits and the protein kinase inhibitor pkiα to inhibit cα phosphotransferase activity in vitro. purified cα was mixed with a fixed concentration of kemptide in the presence of increasing concentrations of the various r subunits or pkiα. all the r subunits inhibited cα2-dependent kinase activity by 50% at nm and showed complete inhibition at a 1: molar ratio. a fixed dose of cα was inhibited by pkiα in a dose-dependent manner with complete inhibition at stoichiometric concentrations of cα and pkiα. the inhibitory effects of the various r subunits and pki have previously been determined for cα and indicate that the efficiency in inhibiting cα and cα is similar for all r subunits and pkiα. using a biacore technology we next investigated the dissociation equilibrium constants, association and dissociation rate constants for the various r subunits in association with either cα or cα we immobilized rus of myristylated cα and cα on separate flow cells of a cm biacore sensor chip. unmyristylated cα was used as reference. in the presence of mm atp and mm mgcl the r subunits were simultaneously run over both c subunits on the sensor chip at a flow rate of μl/min. in the case of riα and riβ they were run over the sensor chip at concentrations between and nm, and riiα and riiβ between to nm. figure shows representative runs of riα, riβ, riiα and riiβ on cα and myrcα the shape of the curves indicates that the relative on and off rates for riα when associated with either cα or myrcα were highly similar. the relative kass values were slightly different, × and × m-1s- for riα versus myrcα and cα respectively. the same was true for riβ. however, in this case, although the kd value was almost identical to riα, the association as well as the dissociation rate constants for riβ was times faster. finally, no differences could be observed for the interaction of riiα and riiβ against myrcα versus cα. in order to investigate the binding behavior of pki, gst-pkiα was immobilized on sensor chips as described previously, and various concentrations of cα cα myrcα and, for comparison, mouse cα were run at a flow rate of μl/min over the sensor chips. this revealed a kd for all c subunits and pkiα at a range around nm where the myrcα displayed a slightly faster association rate compared to cα with all the dissociation rates being similar. spr measurements with single concentrations demonstrated almost identical shapes of the curves, indicating comparable association and dissociation rates. in order to determine accurate association rate constants, different concentrations of the respective c subunits were applied, leading again to the conclusion that the binding activities of cα and cα for pkiα are highly similar. discussion at the protein level cα and cα are 97% homologous and only differ at the n-terminal end. based on this we investigated to what extent differences at the n-terminus may influence splice variant-specific activities that may have biological importance. we found that cα expressed in bacteria was not captured by inclusion bodies as was the case with cα moreover, the specific activity of cα was lower compared to cα apart from these differences we observed that cα was highly similar to cα in all parameters measured. this included association of cα with ri and rii to form camp-sensitive holoenzymes both in vivo and in vitro. furthermore, km values of cα for kemptide and atp were comparable to those determined for cα this was also the case for the ability of ri, rii and pki to inhibit cα enzyme activity in vitro. finally, kd values as measured by spr were shown to be comparable between cα and cα towards the ri and rii subunits as well as pki. several reports imply that n-terminal modifications of cα introduce specific features that may have biological consequences. to this end it has been suggested that phosphorylation of ser in cα introduces an electrostatic force which may help the c subunit to remain soluble even when myristylated. moreover, two reports have demonstrated that the n-terminal myristyl moiety of cα is embedded in a hydrophobic pocket encompassed in the large lobe. mutation of gly to ala rendering the cα non-myristylated, demonstrated that myristylation was non-essential for conformation and enzyme activation, and was not required for cα interaction with other proteins including various substrates and the r subunits. the fact that cα is not myristylated and displays comparable activities with myristylated cα suggests that myristylation is not essential for catalytic activity, holoenzyme formation and inhibition by pki. this is further supported in that deletion of the entire cα n-terminus did not severely interfere with catalytic activity and inhibitor binding despite that deletion caused thermo instability. this also suggests that the amino acids and of cα are not essential for activity a suggestion which is supported by our results on cα cα and cβ which are 100% identical at the n-terminus, but only 91% identical in the sequence encode by exon through have different apparent sizes and possess distinct biochemical properties both in vitro and in vivo. these differences include differential km values for certain peptide substrates and that cα but not cβ is inhibited by substrate concentrations above μm. in addition, they display distinct ic values for pki and riiα. taken together with our results this may suggest that the amino acid sequence encoded by exon through and not exon influence c subunit activities such as holoenzyme formation, enzyme activity and inhibition by r and pki. this may further imply that cα and cβ have distinct roles in regulating cellular processes. this was recently shown in t cells which express cα cβ and cβ in that study cα but none of the cβ forms, mediated the inhibitory effect of camp on immune cell reactivity in vivo. in light of these observations it is also of interest to note that cα but not cα is required for sperm cell forward velocity and male fertility, despite 100% identity at the amino acid sequence encoded by exon through. however, since cα is the sole c subunit in sperm cells, the difference observed may only be ascribed to tissue-specific expression and not sequence-specific differences. in contrast to cα it is expected that the hydrophobic pocket in which the myristyl group is submersed in cα is constitutively empty and exposed to the surroundings at all time in non-myristylated cα it has been speculated whether exposure of the hydrophobic pocket would introduce more lipophilic properties to the cα subunit. support for such a hypothesis is found in a by a study demonstrating that binding of cα to rii induced a unique conformation that is associated with exposure of the hydrophobic pocket to the surroundings due to increase in n-terminal flexibility of the n-myristate away from the large lobe. this renders cα more hydrophobic and promotes membrane association of the pka ii holoenzyme only. therefore it may be suggested that exposure of the hydrophobic pocket serves features such as isoform specific features and subcellular localization of the c subunit. to this end it is interesting to note that cα is associated with the sperm tail in the presence of detergent treatment with 1% triton x-.and, after a challenge with mm camp. this may be indicative of a direct association of cα with subcellular structures. to what extent such attachment involves the hydrophobic pocket remains unknown. in other cells and tissues, c subunits targeted to subcellular structures independent of the r subunit and traditional a-kinase anchoring proteins have been demonstrated. to day a number of c subunit binding proteins have been identified. these include pki, a-kinase interacting protein, homologous to akap, inhibitor of nfkappab kinase, caveolin- and p neutrophine receptor. to what extent cα is targeted to the sperm cell midpiece through a c interaction protein and if specificity of binding is retained in the hyper variable n-terminal end remains to be shown. however, it should be noted that deamination of the asn moiety in cα helps fine-tuning enzyme distribution within the cell in vivo. moreover, p75ntr was shown to specifically bind to the cβ splice variant cβ4ab, which is encoded with unique n-terminal domain that may not undergo the same posttranslational modifications as cα. together this may imply that the n-terminal end may be important for targeting and specificity of subcellular localization of the various c subunits.  our study demonstrates that n-terminal sequence encoded by alternative use of exons upstream of exon in the prkaca gene does not influence c subunit activities such as holoenzyme formation, camp sensitivity, enzyme activity as well as inhibition by ri, rii and pki. based on several studies it may be suggested that the n-terminus is involved in other c subunit features such as subcellular localization. authors' contributions mmv has written the manuscript and has designed an contributed to the experiments in figures through and table and hmz has contributed to the biacore experiments. em has performed the experiments in figures and hvb has made recombinant proteins for cα and cα and contributed to the experiments in figure fwh and bss have conceived the ideas to the manuscript and contributed to the writing. all authors read and approved the final manuscript. abbreviations pka: protein kinase a; c: catalytic subunit; cα: alpha-form of c; cα2: sperm-specific c subunit. 
we show that cα interacts with the two major forms of the regulatory subunit of pka, ri and rii, to form camp-sensitive pkai and pkaii holoenzymes both in vitro and in vivo as is also the case with cα moreover, using surface plasmon resonance, we show that the interaction patterns of the physiological inhibitors ri, rii and pki were comparable for cα and cα this is also the case for their potency to inhibit catalytic activities of cα and cα in cα but not cα the n-terminal end introduces three sites for posttranslational modifications which include myristylation at gly asp-specific deamidation at asn and autophosphorylation at ser previous reports have implicated specific biological features correlating with these modifications on cα since cα is not modified in the same way as cα we tested if they have distinct biochemical activities that may be reflected in different biological properties. the two variants of the α-form of the catalytic subunit of protein kinase a, designated cα and cα are encoded by the prkaca gene. whereas cα is ubiquitous, cα expression is restricted to the sperm cell.
 the two variants of the α-form of the catalytic subunit of protein kinase a, designated cα and cα are encoded by the prkaca gene. whereas cα is ubiquitous, cα expression is restricted to the sperm cell. cα and cα are encoded with different n-terminal domains. in cα but not cα the n-terminal end introduces three sites for posttranslational modifications which include myristylation at gly asp-specific deamidation at asn and autophosphorylation at ser previous reports have implicated specific biological features correlating with these modifications on cα since cα is not modified in the same way as cα we tested if they have distinct biochemical activities that may be reflected in different biological properties.  we show that cα interacts with the two major forms of the regulatory subunit of pka, ri and rii, to form camp-sensitive pkai and pkaii holoenzymes both in vitro and in vivo as is also the case with cα moreover, using surface plasmon resonance, we show that the interaction patterns of the physiological inhibitors ri, rii and pki were comparable for cα and cα this is also the case for their potency to inhibit catalytic activities of cα and cα  we conclude that the regulatory complexes formed with either cα or cα respectively, are indistinguishable. pkacatalytic subunitn-terminalsplice variants 