BACKGROUND
the alpha kinase has been initially identified and characterized in dictyostelium discoideum as myosin heavy chain kinase  <cit> .
unlike many conventional protein kinases, which phosphorylate the protein sites embedded in beta-sheet secondary structures <cit> ; the phosphorylation sites in the substrates of the alpha kinase tended to be in the configuration of an alpha helix <cit> .
the alpha kinase was hypothesized to be the consequence of recent evolution, and believed to play key roles in complex signaling transductions in higher organisms  <cit> .
at present, a total of six alpha kinase members have been identified in the vertebrates, including elongation factor- <dig> kinase , subfamily m member  <dig> and  <dig> of transient receptor potential cation channel , and alpha-kinase 1- <dig>  <cit> .
eukaryotic elongation factor- <dig> kinase  is a ca2+ and calmodulin-dependent kinase <cit> , regulating the global protein translation  <cit> .
the activity of eef2k was also reported to be modulated by the mtor and ampk signaling pathways  <cit> .
trpm <dig> and trpm <dig> have similar protein structures, both consisting of transient receptor potential  cation channels in the n-terminal and alpha-kinase domain in the c-terminal <cit> .
trpm <dig> is important for maintaining whole body mg2+ levels <cit> , while trpm <dig> might be involved in the ca2+ signaling  <cit> .
alpk <dig>  alpk <dig> and alpk <dig> all carry the alpha-kinase domains in the c-terminal <cit> .
alpk <dig> was shown to phosphorylate the myosin ia and play a role in the apical vesicle transport in epithelial cells  <cit> .
the functions of alpk <dig> and alpk <dig> are largely unknown.
piggybac, a dna transposon, was originally found in cabbage looper moth trichoplusiani <cit>  and reported recently as an useful genetic manipulation tool in mice <cit> .
in the present study, we characterized the alpk <dig> insertedmice and found that in a series of behavioral analyses, severe motor coordination deficits were observed in the alpk1pb/pb mice, indicating that alpk <dig> may play an important role in the control of the fine motor activity.
RESULTS
genetic characterization of alpk1pb/pb mutant mice
one pb insertion line, h362cr <dig>  was mapped and found that pb transposon was inserted into the first intron of the alpk <dig> gene .
by using rt-pcr with primer pairs located on exon  <dig> and exon  <dig> of the alpk <dig> gene to amplify the 5'-end transcript, the lack of the intact alpk <dig> transcript was observed in homozygous mice  , indicating that the endogenous alpk <dig> transcript was disrupted by the pb insertion.
to examine the expression level of the alpk <dig> coding sequence, real-time quantitative pcr  was applied to quantify the 3'-end transcripts with primer pairs located on exon  <dig> and exon  <dig> of the alpk <dig> gene.
the transcription level of alpk <dig> was decreased in many tissues, including skeletal muscle , thymus, spleen, lymph node and small intestine , whereas it was increased in the brain .
by using western blot with the rabbit polyclonal antibody specific to the c-terminal of alpk <dig> , we found that alpk <dig> was expressed ubiquitously, consistent with the expression profiling in the mouse microarray analyses http://symatlas.gnf.org/symatlas/.
two protein isoforms were detected with molecular weight of about  <dig> kd and  <dig> kd, which are consistent with the protein sizes predicted by the mouse genome database .
the <dig> kd isoform was highly expressed in the brain, spinal cord, heart, lung, spleen, thymus, small intestine, skin and testis, while detectable in skeletal muscles and kidneys.
the  <dig> kd isoform was found in the heart, lung, thymus and skin.
furthermore, the alpk <dig> protein levels in alpk1pb/pb mice were decreased in most of the tissues except in the brain and the spinal cord, where the protein levels increased , consistent with the results of qpcr.
impaired motor coordinationin alpk1pb/pbmice
compared to the wild type mice, the alpk1pb/pbmutants exhibited elevated tail posture during walking, and occasionally their tails were bent forward .
feet-clasping was observed when the alpk1pb/pb mice were suspended by their tails .
a series of behavioral tests was performed to examine motor control in mutants.
the alpk1pb/pbmice could not stand on the rotating rod on the rotarod test .
in the dowel test, the alpk1pb/pb mice could not easily keep balance on the fixed wooden bar .
the retention time of the alpk1pb/pb micein the hanging wire test was much less than those of the alpk1pb/+ or wild type mice.
in the footprint test, the step width of the alpk1pb/pb mice was wider than that of the control mice  , and the alternation coefficient index also showed significant differences between the alpk1pb/pb mice and the controls  .
all these results implied that the alpk <dig> gene may play an important role in motor coordination.
mating of the alpk1pb/+ mice yielded a near-mendelian distribution of genotypes in the offspring, and gender ratio of offspring was normal.
the survival proportion of the alpk1pb/pb mice was similar to that of wild type controls until they were one year old.
cerebellar morphology and function in alpk1pb/pb mice
since the cerebellum plays an important role in the coordination of movements <cit> , multiple aspects of the cerebellum were examined in alpk1pb/pb mice, including the cerebellar architecture, purkinje cell morphology and the electrophysiological properties of the purkinje cell.
foliation and lamination of the cerebellar cortex appeared normal in alpk1pb/pbmice by histological analysis on cerebellar sections .
immunofluorescence staining with anti-calbindin antibody was performed to examine purkinje cell morphology in details.
the alignment of purkinje cells between the granular and molecular layers , purkinje cell dendritic branches , dendritic length  andpurkinje cell numbers  appeared normal in alpk1pb/pb mice compared tothe wildtype controls.
purkinje cell body in alpk1pb/pb mice was smaller than those of the controls .
the cerebellar long-term depression  did not show significant differences between the alpk1pb/pb mice and the wild type controls .
transgenic rescue of defective motor coordination in alpk1pb/pb mice
hemagglutinin -tagged murine full-length alpk <dig> coding sequence driven by the ubiquitously expressed chicken beta-actin promoter cassette  was constructed to generate transgenic mice .
as the ha tag was fused to the amino-terminal end of alpk <dig>  only the  <dig> kd protein isoform of transgene could be directly detected by using anti-ha antibody in western blot.
indeed, the  <dig> kd isoform of transgene expression was detected in the thymus by anti-ha immunoblotting .
in some tissues, such as the skeletal muscle and brain, alpk <dig> only expressed in  <dig> kd short isoform, excluding the possibility for detection of transgene expression by anti-ha immunoblotting.
therefore, comparison of densitometric immunoreactive intensity in the anti-alpk <dig> immunoblots was utilized to verify transgene expression in those tissues.
the relative prevalence of total alpk <dig> immunoreactivity in skeletal muscle from the alpk1pb/pb mice was  <dig>  ±  <dig>  times than that of wild type controls.
in comparison, the levels of alpk <dig> expression in skeletal muscle from the pcx:haalpk <dig> and the pcx:haalpk1;alpk1pb/pb mice were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  times than wild type controls , indicating that the transgene was highly expressed in skeletal muscle.
in the brain, the relative prevalence of total alpk <dig> immunoreactivity from pcx:haalpk <dig> mice was  <dig>  ±  <dig>  times than that of wild type controls, suggesting that the transgene was expressed in the brain.
the levels of alpk <dig> expression in brain from the alpk1pb/pb and the pcx:haalpk1;alpk1pb/pb mice were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  times than that of wild type controls, respectively .
in the behavioural tests, the performance of pcx:haalpk1;alpk1pb/pb was similar to wild type controls in the dowel test  and in the rotarod test, indicating that the transgenic alpk <dig> could rescue motor coordination deficits in alpk1pb/pb mice.
discussion
alpk <dig>  also known as lymphocyte alpha-kinase, was initially identified in the human lymphocyte cdna library <cit> .
our anti-alpk <dig> immunoblot results confirmed that alpk <dig> was highly expressed in lymphoid organs, such asthymus and spleen, implicating that alpk <dig> might function in the development of the immune system.
moreover, the expression level of alpk <dig> in lymphoid organs was significantly decreased by pb insertion in alpk1pb/pb mice, leading to speculation as to whether the immune system may be affected in mutants.
facs analysis of different markers on cd4+, cd8+ and b cellswere performed, and the proportion of t and b lymphocyte populationsin alpk1pb/pb mice was not changed compared to those of the wild type controls .
further studies may be required to assess whether alpk <dig> plays a role in the immune system.
besides motor coordination deficits, the alpk1pb/pb mice also have other interesting abnormalities.
the alpk1pb/pb mice exhibited mild thoracolumbar kyphosisby micro-ct scanning .
however, further analysis on bone density, bone trabecula, and the structure of sacroiliac joint presented no differences between the alpk1pb/pb and the wild type mice , implying that the kyphosis may be attributed to other causes apart from bone development.
the alpk1pb/pb mice also showed male infertility.
histological analysis revealed that the testes in the alpk1pb/pb mice seemed to develop normally.
sperm derived from alpk1pb/pbor wild type mice was used for in vitro fertilization, and no significant differences in the efficacy of offspring production were observed between the two groups .
when videotaping the sexual behavior in mice , we found that the male alpk1pb/pb mice could not properly mount the female mice, leading to afailure of the mating process.
abnormal mounting ability in the male alpk1pb/pb mice is likely associated with motor coordination deficits in the mutants.
alpk <dig> protein levels were increased in the brain of alpk1pb/pb mice.
however, several lines of evidence went against the hypothesis that motor coordination deficits in the alpk1pb/pb mice may be caused by the increased protein levels in mutant brains.
first, our results showed that two protein isoforms of alpk <dig>  presented in a tissue-dependent manner, while only  <dig> kd isoform could be detected in brains from both alpk1pb/pband the wild type mice.
in order to distinguish the slight differences of alpk <dig> proteins in the alpk1pb/pband wild type mice brains, 2d-page analysis was performed.
one spot with the same migration position could be detected in the gel by our anti-alpk <dig> antibody , indicating that the elevated  <dig> kd isoforms in brains of the alpk1pb/pbmice may be biochemically similar or identical to those produced by the wild type control brains, and pb insertion in the alpk1pb/pbmice might not affect the translation initiation site of alpk1transcript in the brain.
second, similar to the situation seen in the alpk1pb/pbmice, only the  <dig> kd isoforms could be detected in the brain extracts from transgenic mice.
nevertheless, the transgenic line alone did not exhibit the defective motor coordination.
third, multiple aspects of the cerebellumwhich have beenproved to play a key role in motor coordinationwere examined in the alpk1pb/pbmice.
no significant differences were observed between the mutants and the wild type controls, implicating that thedefective motor control in mutants may act in a cerebellum-independent manner.
the change of the expression of alpk <dig> in skeletal muscle was consistent with the appearance/disappearance of the motor coordination deficits in the mice with different genotypes.
however, no obvious differences were observedin the histological analysis of the skeletal musclebetween the wild type and alpk1pb/pbmice .
at present, the mechanisms underlying the severe motor coordination deficits in the alpk1pb/pbmice remain elusive.
tissue-specific transgenic mice would be necessary for further investigations to narrow down the affected tissues in the alpk1pb/pbmice.
CONCLUSIONS
in the present study, mice for alpk1pb/pb alleles were phenotypically characterized and severe motor coordination defects were observed in the alpk1pb/pb mice in multiple behavioral tests.
transgenic mice expressing full-length murine coding sequence of alpk <dig> were capable of rescuing the motor deficits in mutants.
no obvious differences in cerebellar architecture, fine structure and ltd of purkinje cells were found in the alpk1pb/pbmice.
in summary, our alpk <dig> inserted mice provided the first genetic evidence that alpk <dig> may play a crucial role in motor coordination.
the alpk1pb/pb mice provided a valuable tool to elucidate the mechanisms of alpk <dig> in the regulation of motor coordination.

 the alpha kinase has been initially identified and characterized in dictyostelium discoideum as myosin heavy chain kinase. unlike many conventional protein kinases, which phosphorylate the protein sites embedded in beta-sheet secondary structures; the phosphorylation sites in the substrates of the alpha kinase tended to be in the configuration of an alpha helix. the alpha kinase was hypothesized to be the consequence of recent evolution, and believed to play key roles in complex signaling transductions in higher organisms. at present, a total of six alpha kinase members have been identified in the vertebrates, including elongation factor- kinase, subfamily m member and of transient receptor potential cation channel, and alpha-kinase 1-. eukaryotic elongation factor- kinase is a ca2+ and calmodulin-dependent kinase, regulating the global protein translation. the activity of eef2k was also reported to be modulated by the mtor and ampk signaling pathways. trpm and trpm have similar protein structures, both consisting of transient receptor potential cation channels in the n-terminal and alpha-kinase domain in the c-terminal. trpm is important for maintaining whole body mg2+ levels, while trpm might be involved in the ca2+ signaling. alpk alpk and alpk all carry the alpha-kinase domains in the c-terminal. alpk was shown to phosphorylate the myosin ia and play a role in the apical vesicle transport in epithelial cells. the functions of alpk and alpk are largely unknown. piggybac, a dna transposon, was originally found in cabbage looper moth trichoplusiani and reported recently as an useful genetic manipulation tool in mice. in the present study, we characterized the alpk insertedmice and found that in a series of behavioral analyses, severe motor coordination deficits were observed in the alpk1pb/pb mice, indicating that alpk may play an important role in the control of the fine motor activity.  genetic characterization of alpk1pb/pb mutant mice one pb insertion line, h362cr was mapped and found that pb transposon was inserted into the first intron of the alpk gene. by using rt-pcr with primer pairs located on exon and exon of the alpk gene to amplify the 5'-end transcript, the lack of the intact alpk transcript was observed in homozygous mice, indicating that the endogenous alpk transcript was disrupted by the pb insertion. to examine the expression level of the alpk coding sequence, real-time quantitative pcr was applied to quantify the 3'-end transcripts with primer pairs located on exon and exon of the alpk gene. the transcription level of alpk was decreased in many tissues, including skeletal muscle, thymus, spleen, lymph node and small intestine, whereas it was increased in the brain. by using western blot with the rabbit polyclonal antibody specific to the c-terminal of alpk, we found that alpk was expressed ubiquitously, consistent with the expression profiling in the mouse microarray analyses http://symatlas.gnf.org/symatlas/. two protein isoforms were detected with molecular weight of about kd and kd, which are consistent with the protein sizes predicted by the mouse genome database. the kd isoform was highly expressed in the brain, spinal cord, heart, lung, spleen, thymus, small intestine, skin and testis, while detectable in skeletal muscles and kidneys. the kd isoform was found in the heart, lung, thymus and skin. furthermore, the alpk protein levels in alpk1pb/pb mice were decreased in most of the tissues except in the brain and the spinal cord, where the protein levels increased, consistent with the results of qpcr. impaired motor coordinationin alpk1pb/pbmice compared to the wild type mice, the alpk1pb/pbmutants exhibited elevated tail posture during walking, and occasionally their tails were bent forward. feet-clasping was observed when the alpk1pb/pb mice were suspended by their tails. a series of behavioral tests was performed to examine motor control in mutants. the alpk1pb/pbmice could not stand on the rotating rod on the rotarod test. in the dowel test, the alpk1pb/pb mice could not easily keep balance on the fixed wooden bar. the retention time of the alpk1pb/pb micein the hanging wire test was much less than those of the alpk1pb/+ or wild type mice. in the footprint test, the step width of the alpk1pb/pb mice was wider than that of the control mice, and the alternation coefficient index also showed significant differences between the alpk1pb/pb mice and the controls. all these results implied that the alpk gene may play an important role in motor coordination. mating of the alpk1pb/+ mice yielded a near-mendelian distribution of genotypes in the offspring, and gender ratio of offspring was normal. the survival proportion of the alpk1pb/pb mice was similar to that of wild type controls until they were one year old. cerebellar morphology and function in alpk1pb/pb mice since the cerebellum plays an important role in the coordination of movements, multiple aspects of the cerebellum were examined in alpk1pb/pb mice, including the cerebellar architecture, purkinje cell morphology and the electrophysiological properties of the purkinje cell. foliation and lamination of the cerebellar cortex appeared normal in alpk1pb/pbmice by histological analysis on cerebellar sections. immunofluorescence staining with anti-calbindin antibody was performed to examine purkinje cell morphology in details. the alignment of purkinje cells between the granular and molecular layers, purkinje cell dendritic branches, dendritic length andpurkinje cell numbers appeared normal in alpk1pb/pb mice compared tothe wildtype controls. purkinje cell body in alpk1pb/pb mice was smaller than those of the controls. the cerebellar long-term depression did not show significant differences between the alpk1pb/pb mice and the wild type controls. transgenic rescue of defective motor coordination in alpk1pb/pb mice hemagglutinin -tagged murine full-length alpk coding sequence driven by the ubiquitously expressed chicken beta-actin promoter cassette was constructed to generate transgenic mice. as the ha tag was fused to the amino-terminal end of alpk only the kd protein isoform of transgene could be directly detected by using anti-ha antibody in western blot. indeed, the kd isoform of transgene expression was detected in the thymus by anti-ha immunoblotting. in some tissues, such as the skeletal muscle and brain, alpk only expressed in kd short isoform, excluding the possibility for detection of transgene expression by anti-ha immunoblotting. therefore, comparison of densitometric immunoreactive intensity in the anti-alpk immunoblots was utilized to verify transgene expression in those tissues. the relative prevalence of total alpk immunoreactivity in skeletal muscle from the alpk1pb/pb mice was ± times than that of wild type controls. in comparison, the levels of alpk expression in skeletal muscle from the pcx:haalpk and the pcx:haalpk1;alpk1pb/pb mice were ± and ± times than wild type controls, indicating that the transgene was highly expressed in skeletal muscle. in the brain, the relative prevalence of total alpk immunoreactivity from pcx:haalpk mice was ± times than that of wild type controls, suggesting that the transgene was expressed in the brain. the levels of alpk expression in brain from the alpk1pb/pb and the pcx:haalpk1;alpk1pb/pb mice were ± and ± times than that of wild type controls, respectively. in the behavioural tests, the performance of pcx:haalpk1;alpk1pb/pb was similar to wild type controls in the dowel test and in the rotarod test, indicating that the transgenic alpk could rescue motor coordination deficits in alpk1pb/pb mice. discussion alpk also known as lymphocyte alpha-kinase, was initially identified in the human lymphocyte cdna library. our anti-alpk immunoblot results confirmed that alpk was highly expressed in lymphoid organs, such asthymus and spleen, implicating that alpk might function in the development of the immune system. moreover, the expression level of alpk in lymphoid organs was significantly decreased by pb insertion in alpk1pb/pb mice, leading to speculation as to whether the immune system may be affected in mutants. facs analysis of different markers on cd4+, cd8+ and b cellswere performed, and the proportion of t and b lymphocyte populationsin alpk1pb/pb mice was not changed compared to those of the wild type controls. further studies may be required to assess whether alpk plays a role in the immune system. besides motor coordination deficits, the alpk1pb/pb mice also have other interesting abnormalities. the alpk1pb/pb mice exhibited mild thoracolumbar kyphosisby micro-ct scanning. however, further analysis on bone density, bone trabecula, and the structure of sacroiliac joint presented no differences between the alpk1pb/pb and the wild type mice, implying that the kyphosis may be attributed to other causes apart from bone development. the alpk1pb/pb mice also showed male infertility. histological analysis revealed that the testes in the alpk1pb/pb mice seemed to develop normally. sperm derived from alpk1pb/pbor wild type mice was used for in vitro fertilization, and no significant differences in the efficacy of offspring production were observed between the two groups. when videotaping the sexual behavior in mice, we found that the male alpk1pb/pb mice could not properly mount the female mice, leading to afailure of the mating process. abnormal mounting ability in the male alpk1pb/pb mice is likely associated with motor coordination deficits in the mutants. alpk protein levels were increased in the brain of alpk1pb/pb mice. however, several lines of evidence went against the hypothesis that motor coordination deficits in the alpk1pb/pb mice may be caused by the increased protein levels in mutant brains. first, our results showed that two protein isoforms of alpk presented in a tissue-dependent manner, while only kd isoform could be detected in brains from both alpk1pb/pband the wild type mice. in order to distinguish the slight differences of alpk proteins in the alpk1pb/pband wild type mice brains, 2d-page analysis was performed. one spot with the same migration position could be detected in the gel by our anti-alpk antibody, indicating that the elevated kd isoforms in brains of the alpk1pb/pbmice may be biochemically similar or identical to those produced by the wild type control brains, and pb insertion in the alpk1pb/pbmice might not affect the translation initiation site of alpk1transcript in the brain. second, similar to the situation seen in the alpk1pb/pbmice, only the kd isoforms could be detected in the brain extracts from transgenic mice. nevertheless, the transgenic line alone did not exhibit the defective motor coordination. third, multiple aspects of the cerebellumwhich have beenproved to play a key role in motor coordinationwere examined in the alpk1pb/pbmice. no significant differences were observed between the mutants and the wild type controls, implicating that thedefective motor control in mutants may act in a cerebellum-independent manner. the change of the expression of alpk in skeletal muscle was consistent with the appearance/disappearance of the motor coordination deficits in the mice with different genotypes. however, no obvious differences were observedin the histological analysis of the skeletal musclebetween the wild type and alpk1pb/pbmice. at present, the mechanisms underlying the severe motor coordination deficits in the alpk1pb/pbmice remain elusive. tissue-specific transgenic mice would be necessary for further investigations to narrow down the affected tissues in the alpk1pb/pbmice.  in the present study, mice for alpk1pb/pb alleles were phenotypically characterized and severe motor coordination defects were observed in the alpk1pb/pb mice in multiple behavioral tests. transgenic mice expressing full-length murine coding sequence of alpk were capable of rescuing the motor deficits in mutants. no obvious differences in cerebellar architecture, fine structure and ltd of purkinje cells were found in the alpk1pb/pbmice. in summary, our alpk inserted mice provided the first genetic evidence that alpk may play a crucial role in motor coordination. the alpk1pb/pb mice provided a valuable tool to elucidate the mechanisms of alpk in the regulation of motor coordination. 
our results indicate that alpk plays an important role in the regulation of motor coordination. the transposon-inserted alpk homozygous mutants displayed severe defects in motor coordination in a series of behavioral analysis, including dowel test, hanging wire test, rotarod analysis and footprint analysis. the motor coordination deficits in the alpk1pb/pb mice were rescued by transgenic mice expressing the full-length alpk1-coding sequence under the control of the ubiquitous expression promoter. however, the cerebellar architecture, purkinje cell morphology and electrophysiology of the purkinje cells appeared normal in mutants. alpk1pb/pb mice would be a useful model to provide a clue to the better understanding of the cellular and molecular mechanisms of alpk in the control of fine motor activities.
 alpk is a member of an unconventional alpha-kinase family, and its biological function remains largely unknown. here we report the phenotypic characterization of one mutant line, in which the piggybac transposon is inserted into the alpk gene.  the piggybac insertion site in mutants was mapped to the first intron of the alpk gene, resulting in the effective disruption of the intact alpk transcript expression. the transposon-inserted alpk homozygous mutants displayed severe defects in motor coordination in a series of behavioral analysis, including dowel test, hanging wire test, rotarod analysis and footprint analysis. however, the cerebellar architecture, purkinje cell morphology and electrophysiology of the purkinje cells appeared normal in mutants. the motor coordination deficits in the alpk1pb/pb mice were rescued by transgenic mice expressing the full-length alpk1-coding sequence under the control of the ubiquitous expression promoter.  our results indicate that alpk plays an important role in the regulation of motor coordination. alpk1pb/pb mice would be a useful model to provide a clue to the better understanding of the cellular and molecular mechanisms of alpk in the control of fine motor activities. 