BACKGROUND
pim- <dig>   <dig> and  <dig> make up a group of closely related mammalian serine/threonine kinases that form part of the calmodulin dependent protein kinase family  <cit> .
pim- <dig> was first identified as an oncogenic gene in murine t-cell lymphomas, which became activated after pro-viral insertion in the 3'utr of the pim- <dig> gene  <cit> .
insertion in this region stabilised the mrna for pim- <dig> thus up-regulating the protein  <cit> .
over-expression of the pim- <dig> gene in b and t cells in transgenic mice resulted in a high level of lymphoma development in these mice, confirming the oncogenic properties of this protein  <cit> .
a second related gene, pim- <dig>  was also identified as a common site of viral insertion in murine lymphomas  <cit> .
like pim- <dig> the over-expression of pim- <dig> in transgenic mice predisposes the mice to the development of lymphoma  <cit> .
the precise mechanism by which pim- <dig> and  <dig> promote the formation of lymphomas is not fully understood, however it has been suggested that pim- <dig> synergises with c-myc in this process  <cit> .
pim- <dig> also appears to be important in the development of human cancers, as the pim- <dig> gene maps to an area showing karyotypic abnormalities in leukaemia, and the mrna for pim- <dig> has been shown to be up-regulated in prostate cancer and leukaemia  <cit> .
a third isoform, pim- <dig>  which is 72% identical to pim- <dig> also exists in mammalian cells although the oncogenic properties of this protein are less well studied.
pim- <dig> has recently been shown to be up-regulated in ewing's tumour cell lines, is a transcriptional target for the oncogenic ews transcription factors and is able to promote anchorage-independent cell growth in tissue culture models  <cit> .
recently, over-expression of pim- <dig> has been demonstrated in a mouse model of hepatocellular carcinoma, although the functional role of pim- <dig> was not determined  <cit> .
several substrates have been suggested for pim- <dig> including mdm <dig>  <cit> , p <dig>  <cit> , cdc <dig> phosphatase  <cit>  and c-myb  <cit> , all proteins involved in control of the cell cycle.
pim- <dig> has been reported to maintain phosphorylation of the translational repressor 4ebp- <dig> and to phosphorylate and inactivate the pro-apoptotic protein bad on ser <dig>  <cit> .
pim- <dig> has also been reported to be able to phosphorylate bad on ser <dig>  <cit> , however the ability of pim- <dig> to phosphorylate bad is unknown.
both pim- <dig> and pim- <dig> therefore appear to act as survival or proliferation signals in vivo.
consistent with this pim- <dig> expression has been shown to promote cytokine independent growth of haematopoetic cells  <cit> .
in contrast, less is known about the substrates of pim- <dig>  and its function in vivo is still largely unclear.
bad is a pro-apoptotic member of the bh <dig> family of proteins.
active bad induces apoptosis by binding to anti-apoptotic bcl- <dig> family members such as bcl-xl.
this allows other pro-apoptotic proteins such as bax and bak to aggregate, inducing cytochrome c release from the mitochondria and caspase activation .
the activity of bad is repressed by phosphorylation, and three residues  have been identified as the predominant in vivo phosphorylation sites.
in addition to pim- <dig> several kinases have been identified as potential kinases for bad, including pkb  <cit> , rsk  <cit> , pak  <cit> , p70s6k  <cit>  and pka  <cit> .
it seems likely that multiple kinases may phosphorylate bad on these sites in vivo, depending on the cell type and situation.
consistent with this, several of the proposed kinases preferentially phosphorylate one or two of the three sites in bad.
for instance, in vitro rsk phosphorylates ser <dig> and to a lesser extent ser <dig>  while pkb phosphorylates ser <dig> and pka preferentially phosphorylates ser <dig>  <cit> .
in addition to these phosphorylation sites, an unidentified kinase has been shown to inhibit apoptosis by phosphorylating bad on ser <dig>  <cit> .
conversely, phosphorylation by cdc <dig> of ser <dig> in bad was found to promote the apoptotic activity of bad  <cit> .
in this paper we examine the specificity of pim- <dig>   <dig> and  <dig> for the ser <dig>   <dig> and  <dig> phosphorylation sites in bad, both in vitro and in hek- <dig> cells.
RESULTS
pim kinases phosphorylate bad on three sites in vitro
bad has been described previously to be phosphorylated on three sites, ser <dig>  ser <dig> and ser <dig> in vivo.
peptides from around these sites were used as substrates for purified pim- <dig>   <dig> or  <dig> in in vitro kinase assays.
all three pim kinases were found to phosphorylate the ser <dig> peptide efficiently .
in contrast the ser <dig> and ser <dig> peptides were much less effective substrates for pim- <dig> and pim- <dig>  and very poor substrates for pim- <dig> .
as phosphorylation of synthetic peptides does not always give a true reflection of a kinases ability to phosphorylate the corresponding site in a protein, the ability of pim kinases to phosphorylate recombinant bad protein was also examined.
as expected bad was phosphorylated by all three pim kinases, with maximum phosphorylation occurring in the first  <dig> to  <dig> min .
this activity was due to the pim kinase, as incubation of the bad protein in the absence of pim , or with kinase dead pim , did not result in any incorporation of 32p into bad.
to determine the sites phosphorylated by pim- <dig>   <dig> or  <dig>  recombinant bad was phosphorylated with 32p labelled atp and digested with trypsin.
peptides were then resolved by reverse phase hplc and ms/ms used to identify the phospho peptides eluted .
the position of the phosphate in each peptide was also confirmed by solid phase sequencing reactions and measuring the cycle at which radioactivity was released.
pim- <dig> phosphorylated  <dig> major peptides in bad, designated p <dig>  p <dig> p <dig> and p <dig> .
peak p <dig> was found to correspond to phospho ser <dig>  p <dig> to ser <dig> while both p <dig> and p <dig> corresponded to phospho ser <dig> .
the different elution times of the ser <dig> peptides is probably explained by different oxidation states of the methionine residue in the peptide.
analysis of the cpm in each peak showed that ser <dig> was the site most highly phosphorylated by pim- <dig>  with approximately  <dig> times the level of phosphorylation than occurred at either ser <dig> or ser <dig>  this was consistent with what was found for the rate of phosphorylation of the synthetic peptides for each site .
pim- <dig> was found to phosphorylate two major peaks that eluted at an identical position to the p <dig> and p <dig> peaks in the pim- <dig> phosphorylated bad .
ms/ms analysis confirmed that these peptides also corresponded to bad phosphorylated on ser <dig>  small peaks were also seen corresponding to p <dig> and p <dig> in the pim- <dig> phosphorylated bad hplc trace, and these were confirmed to correspond to phospho ser <dig> and ser <dig>  the amount of phosphate incorporated at these sites was  <dig> times less than that at ser <dig> .
pim- <dig> phosphorylated bad also gave peaks corresponding to p <dig>  p <dig>  p <dig> and p <dig>  and again these were confirmed as ser <dig>  ser <dig> and ser <dig> by ms/ms and solid phase sequencing .
of the three kinases pim- <dig> gave the highest phosphorylation of ser <dig> and ser <dig> compared to ser <dig> in bad .
five further smaller phospho peptide peaks  were also observed in the hplc when pim- <dig> was used to phosphorylate bad.
these sites were not however significantly phosphorylated by pim- <dig> or pim- <dig>  p <dig> was observed in the flow through of the column and peptides in this fraction were not identified by mass spectrometry.
prediction from the bad sequence shows that tryptic digest would give  <dig> small peptides that contain serine or threonine that could have been phosphorylated.
phosphopeptides from p <dig> were also not successfully identified by mass spectrometry.
p <dig> was identified as the peptide aatnshhggagametr  with  <dig> phospho group.
ms/ms analysis suggested that this was a mixture of phosphorylation on either thr <dig> or ser <dig>  solid phase sequencing confirmed this result, and also suggested that ser <dig> was the predominant site .
p <dig> and p <dig> were identified by mass spectrometry as sagtatqmr  phosphorylated at ser <dig>  the phosphorylation of ser <dig> was confirmed by solid phase sequencing for both p <dig> and p <dig>  surprisingly, solid phase sequencing also showed that in p <dig> the 4th residue in the peptide was also phosphorylated, suggesting that thr  <dig> may also be phosphorylated by pim- <dig>
pim kinases are constitutively active in hek- <dig> cells and phosphorylate bad on ser <dig>   <dig> and 155
to examine the regulation of pim- <dig>   <dig> and  <dig> in cells, flag tagged pim kinases were expressed in hek- <dig> cells by transient transfection.
endogenous levels of pim- <dig>   <dig> or  <dig> could not be detected in hek- <dig> cells with the antibodies available.
after serum starvation the cells well treated with various stimuli and the activity of the pim kinases determined after immunoprecipitation with an anti-flag antibody.
all three of the pim kinases were found to be active in serum starved cells, and their activity was not further increased by stimulation of the cells with mitogenic signals , cellular stress  or aicar, an activator of ampk .
gst-bad expressed by transient transfection of serum starved hek- <dig> cells, in the absence of the co-expression of any kinase, was found to be phosphorylated on ser <dig>  bur not ser <dig> or ser <dig>  as judged by immunoblotting using antibodies specific for the various phosphorylation sites.
consistent with previous studies  <cit> , stimulation of the cells with pma resulted in a modest increase in ser <dig> phosphorylation whilst forskolin resulted in increases in ser <dig> phosphorylation and, to a lesser extent, ser <dig> .
co-expression of pim- <dig> and bad resulted in increased phosphorylation of ser <dig> and phosphorylation of ser <dig> and ser <dig>  when compared to bad expressed in the absence of pim- <dig>  similar results were also obtained for co-expression of pim- <dig> or pim- <dig>  although pim- <dig> had a reduced effect on ser <dig> phosphorylation in bad compared to co-expression with pim- <dig> and pim- <dig> .
the phosphorylation of bad by pim kinases was not further increased by pma stimulation, although ser <dig> phosphorylation was further increased by forskolin stimulation.
the phosphorylation of bad was dependent on the kinase activity of pim kinases, as co-expression of kinase dead versions of pim- <dig>   <dig> or  <dig> and bad did not result in the phosphorylation of bad .
the ability of pim- <dig> to phosphorylate bad in which one of the phosphorylation sites had been mutated was also determined by co-expression.
as expected the mutation of ser <dig> to alanine prevented phosphorylation of this site by pim- <dig>  but did not significantly affect the phosphorylation of either ser <dig> or ser <dig> .
similarly, mutation of ser <dig> prevented phosphorylation of this site by pim- <dig> but did not significantly affect phosphorylation of ser <dig>  surprisingly mutation of ser <dig> to alanine did cause a reduction in the level of ser <dig> phosphorylation by pim- <dig>  and a similar reduction in ser <dig> phosphorylation was also seen in ser112/ser155ala mutants .
mutation of ser <dig> to alanine not only blocked phosphorylation of ser <dig> by pim- <dig>  but also greatly reduced ser <dig> and ser <dig> phosphorylation .
mutation of ser <dig> to an acidic residue to mimic phosphorylation was able to restore phosphorylation of ser <dig> and ser <dig> .
similar effects of the mutants were also seen in the absence of co-expressed pim- <dig>  when cells were stimulated with agents to induce the phosphorylation of specific sites within bad.
pma promotes bad ser <dig> phosphorylation, probably via rsk, however this was prevented by ser <dig> mutation.
forskolin stimulated ser <dig> phosphorylation via pka, and this was also prevented by mutation of ser <dig> .
pim promotes 14-3- <dig> binding to bad via phosphorylation of ser <dig> and dissociation from bcl-xl
phosphorylation of a protein can create binding sites for 14-3- <dig> proteins, and ser <dig> and  <dig> in bad lie in a potential consensus 14-3- <dig> motif  <cit> .
ser <dig> does not lie in a consensus 14-3- <dig> binding motif, however in vitro phosphorylation of bad by pka  does promote 14-3- <dig> interaction  <cit> .
to test the ability of bad to interact with 14-3- <dig>  gst-bad was co-expressed in hek- <dig> cells with pim- <dig>  and 14-3- <dig> overlay assays were used to determine if bad could bind 14-3- <dig> proteins.
in the overlay assay, recombinant 14-3- <dig> protein was used to detect the presence of proteins on a western blot capable of binding 14-3- <dig>  gst-bad isolated by glutathione-sepharose pull downs from the cell lysates was tested for its ability to bind to 14-3- <dig>  in the absence of pim- <dig> co-expression, gst-bad was not significantly phosphorylated on ser <dig>   <dig> or  <dig> and did not bind 14-3- <dig> in the overlay assay .
co-expression of pim- <dig> however resulted in phosphorylation of all three sites and promoted interaction of the gst-bad with 14-3- <dig> in the overlay assay.
mutation of either ser <dig> or ser <dig> to alanine resulted in a modest reduction in the ability of the pim- <dig> phosphorylated gst-bad to interact with 14-3- <dig> proteins in the overlay assay .
as expected, mutation of ser <dig> to alanine, which also blocks the phosphorylation of ser <dig> and ser <dig>  completely blocked the ability of gst-bad to interact with 14-3- <dig> proteins.
mutation of ser <dig> to an acidic residue also blocked the ability of bad to bind to 14-3- <dig> proteins in the overlay assay, even though pim- <dig> was able to phosphorylate both ser <dig> and ser <dig> in the ser136asp or ser136glu bad mutants .
these data suggest that ser <dig> is the major 14-3- <dig> binding site.
consistent with this, a double mutant of both ser <dig> and ser <dig> to alanine, which was phosphorylated on ser <dig> , was still able to interact with 14-3- <dig> proteins in the overlay assay .
similar to pim- <dig>  when pim- <dig> or pim- <dig> were co-expressed with gst-bad, they were also able to promote 14-3- <dig> binding in the overlay assay .
to test if pim kinases promoted the interaction of bad and 14-3- <dig> proteins in cells, pim kinases and gst-bad were co-expressed in hek- <dig> cells and the gst-bad isolated by glutathione-sepharose pull downs and blotted for the presence of endogenous 14-3- <dig> proteins using a pan 14-3- <dig> isoform antibody .
in the absence of pim kinase co-expression, 14-3- <dig> proteins were not detected in the gst-bad pull downs, however expression of pim- <dig>   <dig> or  <dig> was found to promote the binding of 14-3- <dig> to gst-bad in cells .
the ability of pim kinases to promote the interaction of bad and 14-3- <dig> was dependent on their kinase activity, as co-expression of kinase dead pim- <dig>   <dig> or  <dig> did not promote 14-3- <dig> binding as judged by either co-precipitation or 14-3- <dig> overlay assays .
phosphorylation of bad has been shown to prevent an interaction with bcl-xl  <cit> .
the ability of pim kinases to prevent the interaction of gst-bad with endogenous bcl-xl was tested in pull down experiments.
hela cells were used for these experiments as these cells express detectable levels of endogenous bcl-xl.
as expected, gst-bad, which was not significantly phosphorylated in the absence of kinase co-expression, was able to interact with bcl-xl in hela cells.
co-expression of pim- <dig>   <dig> or  <dig> resulted in phosphorylation of gst-bad and reduced the interaction of gst-bad with bcl-xl.
this was dependent on the kinase activity of pim, as co-expression of kinase dead pim mutants did not have this effect .
similar to the 14-3- <dig> interaction, ser <dig> appeared to be the critical residue involved in this process.
mutation of ser <dig>  ser <dig> , or both ser <dig> and ser <dig> to alanine  in bad did not affect the ability of pim- <dig> to prevent the interaction of gst-bad and bcl-xl.
mutation of ser <dig> in bad to alanine  or an acidic residue  blocked the ability of pim- <dig> to promote the dissociation of gst-bad and bcl-xl .
discussion
pim- <dig>   <dig> or  <dig> were found to be able to phosphorylate bad in vitro and in cells.
pim- <dig> was found to be the most specific of the pim kinases for ser <dig> of bad in vitro, although it was also able to phosphorylate ser <dig> both in vitro and when over-expressed in hek- <dig> cells.
pim- <dig> was a poor ser <dig> kinase in vitro and, consistent with this, was the least effective of all the pim kinases at phosphorylating this site in cells.
in agreement with this pim- <dig> has previously been described to phosphorylate ser <dig>  <cit> .
interestingly, the phosphorylation of ser <dig> and ser <dig> by over-expression of pim- <dig> has been suggested to be dependent on the cell line used  <cit> .
both previous results and our results suggest this may reflect different over-expression levels of pim- <dig> achieved in the different cell types used.
pim- <dig> was able to phosphorylate ser <dig>  ser <dig> and ser <dig> in bad, however phosphorylation of ser <dig> was significantly more efficient than phosphorylation of the other sites in vitro.
pim- <dig> has previously been found to phosphorylate ser <dig> in bad, although its ability to phosphorylate bad on other sites has not previously been reported  <cit> .
the ability of pim- <dig> to phosphorylate bad has not previously been addressed.
here we report that pim- <dig> is also able to phosphorylate ser <dig> in bad.
interestingly pim- <dig> was the most effective kinase in vitro for ser <dig> of bad.
unlike pim- <dig> or pim- <dig>  which demonstrated a marked preference for ser <dig>  pim- <dig> was also effective at phosphorylating ser <dig>  and to a lesser extent ser <dig>  when bad protein was used as an in vitro substrate .
in addition pim- <dig> was also able to phosphorylate other sites in bad including ser <dig> in vitro.
this site has been reported to be phosphorylated in vivo, although the kinase responsible was not identified in the initial study  <cit> .
mutagenesis of ser <dig> in bad has suggested that phosphorylation of this site, like phosphorylation of ser <dig>   <dig> and  <dig>  inhibits the pro-apoptotic activity of bad  <cit> .
phosphorylation of this site by pim- <dig> would therefore be consistent with the pro-survival function of pim- <dig>  to the best of our knowledge, this study is the first to report a pim- <dig> cellular substrate.
the interaction between the phosphorylation sites in bad and their roles in bad function is complex.
phosphorylation of ser <dig> appears to be required for the phosphorylation of ser <dig> and ser <dig> to occur when bad is over-expressed in cells.
similarly, ser <dig> appears to be the major 14-3- <dig> binding site in bad   <cit> , while ser <dig> is suggested to be required for binding to bcl-xl  <cit> .
despite this, phosphorylation of endogenous bad on ser <dig> has proven hard to demonstrate in vivo.
this may reflect the poor sensitivity of the antibodies available, however it has also been suggested that this site may not be physiologically important  <cit> .
in contrast to this, studies using transfected bad have suggested that phosphorylation of this site is essential for the phosphorylation of ser <dig> and ser <dig> and 14-3- <dig> binding, which are required to inhibit the apoptotic activity of bad  <cit> .
studies on ser <dig> have suggested that this site may be a 'gatekeeper' site, and that dephosphorylation of this site is required to recruit a phosphatase that then dephosphorylates ser <dig> and ser <dig>  <cit> .
an extension of this idea lead to the proposal that pim- <dig> only directly phosphorylates ser <dig>  and that an increase in ser <dig> phosphorylation when pim- <dig> was over-expressed was due to a decrease in the rate of ser <dig> dephosphorylation  <cit> .
whilst this cannot be completely excluded, it is not consistent with our data that pim- <dig> can promote the phosphorylation of a ser112ala bad mutant in cells.
the interplay between the different bad phosphorylation sites is thus very complex and further work is required to completely understand these mechanisms.
an added degree of complexity is the number of physiological kinases for bad, including pim-1- <dig>  pkb, pak, rsk, pka  <cit>  and possibly other kinases potentially playing a role.
the exact context in which pim phosphorylates bad in vivo may therefore depend on both cell type and stimulus and remains to be fully understood.
the ability of pim kinases to phosphorylate bad is consistent with the suggested role of these kinases in pro survival or proliferation pathways.
the finding that all three pim kinases can phosphorylate similar sites in bad could in part explain the redundancy between pim kinases in providing a cellular survival signal.
the knockout of pim kinase might therefore be expected to have a severe phenotype, however a triple knockout of pim- <dig>   <dig> and  <dig> has recently been reported.
surprisingly these mice were found to be viable, and while they were smaller in size than wild type mice they displayed no obvious health problems  <cit> .
these mice did however show reduced proliferation of some haematopoietic cells in response to growth factors.
interestingly, a triple knockin mutation of bad  in mice was also found to be viable and did not show increased levels of apoptosis in tissues under normal conditions  <cit> .
these mice also displayed reduced numbers of t and b cells and reduced cytokine induced survival of these cells in vitro, a phenotype with some similarity to that of the triple pim kinase knockout.
CONCLUSIONS
we present here biochemical evidence that all three isoforms of pim kinase are able to phosphorylate bad.
the major site of bad phosphorylation for all three isoforms of pim kinase is ser <dig>  however, we also demonstrate that pim kinases are able to phosphorylate bad to a lesser extent on ser <dig> and ser <dig> in vitro.
interestingly pim- <dig> exhibits less specificity for ser <dig> than pim- <dig> or pim- <dig>  and was the most effective pim isoform for ser <dig> and ser <dig> phosphorylation in bad in vitro.
we also provide evidence that pim kinases may modulate the phosphorylation of bad on ser <dig>  ser <dig> and ser <dig> in hek- <dig> cells.

 pim- and make up a group of closely related mammalian serine/threonine kinases that form part of the calmodulin dependent protein kinase family. pim- was first identified as an oncogenic gene in murine t-cell lymphomas, which became activated after pro-viral insertion in the 3'utr of the pim- gene. insertion in this region stabilised the mrna for pim- thus up-regulating the protein. over-expression of the pim- gene in b and t cells in transgenic mice resulted in a high level of lymphoma development in these mice, confirming the oncogenic properties of this protein. a second related gene, pim- was also identified as a common site of viral insertion in murine lymphomas. like pim- the over-expression of pim- in transgenic mice predisposes the mice to the development of lymphoma. the precise mechanism by which pim- and promote the formation of lymphomas is not fully understood, however it has been suggested that pim- synergises with c-myc in this process. pim- also appears to be important in the development of human cancers, as the pim- gene maps to an area showing karyotypic abnormalities in leukaemia, and the mrna for pim- has been shown to be up-regulated in prostate cancer and leukaemia. a third isoform, pim- which is 72% identical to pim- also exists in mammalian cells although the oncogenic properties of this protein are less well studied. pim- has recently been shown to be up-regulated in ewing's tumour cell lines, is a transcriptional target for the oncogenic ews transcription factors and is able to promote anchorage-independent cell growth in tissue culture models. recently, over-expression of pim- has been demonstrated in a mouse model of hepatocellular carcinoma, although the functional role of pim- was not determined. several substrates have been suggested for pim- including mdm, p, cdc phosphatase and c-myb, all proteins involved in control of the cell cycle. pim- has been reported to maintain phosphorylation of the translational repressor 4ebp- and to phosphorylate and inactivate the pro-apoptotic protein bad on ser. pim- has also been reported to be able to phosphorylate bad on ser, however the ability of pim- to phosphorylate bad is unknown. both pim- and pim- therefore appear to act as survival or proliferation signals in vivo. consistent with this pim- expression has been shown to promote cytokine independent growth of haematopoetic cells. in contrast, less is known about the substrates of pim- and its function in vivo is still largely unclear. bad is a pro-apoptotic member of the bh family of proteins. active bad induces apoptosis by binding to anti-apoptotic bcl- family members such as bcl-xl. this allows other pro-apoptotic proteins such as bax and bak to aggregate, inducing cytochrome c release from the mitochondria and caspase activation. the activity of bad is repressed by phosphorylation, and three residues have been identified as the predominant in vivo phosphorylation sites. in addition to pim- several kinases have been identified as potential kinases for bad, including pkb, rsk, pak, p70s6k and pka. it seems likely that multiple kinases may phosphorylate bad on these sites in vivo, depending on the cell type and situation. consistent with this, several of the proposed kinases preferentially phosphorylate one or two of the three sites in bad. for instance, in vitro rsk phosphorylates ser and to a lesser extent ser while pkb phosphorylates ser and pka preferentially phosphorylates ser. in addition to these phosphorylation sites, an unidentified kinase has been shown to inhibit apoptosis by phosphorylating bad on ser. conversely, phosphorylation by cdc of ser in bad was found to promote the apoptotic activity of bad. in this paper we examine the specificity of pim- and for the ser and phosphorylation sites in bad, both in vitro and in hek- cells.  pim kinases phosphorylate bad on three sites in vitro bad has been described previously to be phosphorylated on three sites, ser ser and ser in vivo. peptides from around these sites were used as substrates for purified pim- or in in vitro kinase assays. all three pim kinases were found to phosphorylate the ser peptide efficiently. in contrast the ser and ser peptides were much less effective substrates for pim- and pim- and very poor substrates for pim-. as phosphorylation of synthetic peptides does not always give a true reflection of a kinases ability to phosphorylate the corresponding site in a protein, the ability of pim kinases to phosphorylate recombinant bad protein was also examined. as expected bad was phosphorylated by all three pim kinases, with maximum phosphorylation occurring in the first to min. this activity was due to the pim kinase, as incubation of the bad protein in the absence of pim, or with kinase dead pim, did not result in any incorporation of 32p into bad. to determine the sites phosphorylated by pim- or recombinant bad was phosphorylated with 32p labelled atp and digested with trypsin. peptides were then resolved by reverse phase hplc and ms/ms used to identify the phospho peptides eluted. the position of the phosphate in each peptide was also confirmed by solid phase sequencing reactions and measuring the cycle at which radioactivity was released. pim- phosphorylated major peptides in bad, designated p p p and p. peak p was found to correspond to phospho ser p to ser while both p and p corresponded to phospho ser. the different elution times of the ser peptides is probably explained by different oxidation states of the methionine residue in the peptide. analysis of the cpm in each peak showed that ser was the site most highly phosphorylated by pim- with approximately times the level of phosphorylation than occurred at either ser or ser this was consistent with what was found for the rate of phosphorylation of the synthetic peptides for each site. pim- was found to phosphorylate two major peaks that eluted at an identical position to the p and p peaks in the pim- phosphorylated bad. ms/ms analysis confirmed that these peptides also corresponded to bad phosphorylated on ser small peaks were also seen corresponding to p and p in the pim- phosphorylated bad hplc trace, and these were confirmed to correspond to phospho ser and ser the amount of phosphate incorporated at these sites was times less than that at ser. pim- phosphorylated bad also gave peaks corresponding to p p p and p and again these were confirmed as ser ser and ser by ms/ms and solid phase sequencing. of the three kinases pim- gave the highest phosphorylation of ser and ser compared to ser in bad. five further smaller phospho peptide peaks were also observed in the hplc when pim- was used to phosphorylate bad. these sites were not however significantly phosphorylated by pim- or pim- p was observed in the flow through of the column and peptides in this fraction were not identified by mass spectrometry. prediction from the bad sequence shows that tryptic digest would give small peptides that contain serine or threonine that could have been phosphorylated. phosphopeptides from p were also not successfully identified by mass spectrometry. p was identified as the peptide aatnshhggagametr with phospho group. ms/ms analysis suggested that this was a mixture of phosphorylation on either thr or ser solid phase sequencing confirmed this result, and also suggested that ser was the predominant site. p and p were identified by mass spectrometry as sagtatqmr phosphorylated at ser the phosphorylation of ser was confirmed by solid phase sequencing for both p and p surprisingly, solid phase sequencing also showed that in p the 4th residue in the peptide was also phosphorylated, suggesting that thr may also be phosphorylated by pim- pim kinases are constitutively active in hek- cells and phosphorylate bad on ser and 155 to examine the regulation of pim- and in cells, flag tagged pim kinases were expressed in hek- cells by transient transfection. endogenous levels of pim- or could not be detected in hek- cells with the antibodies available. after serum starvation the cells well treated with various stimuli and the activity of the pim kinases determined after immunoprecipitation with an anti-flag antibody. all three of the pim kinases were found to be active in serum starved cells, and their activity was not further increased by stimulation of the cells with mitogenic signals, cellular stress or aicar, an activator of ampk. gst-bad expressed by transient transfection of serum starved hek- cells, in the absence of the co-expression of any kinase, was found to be phosphorylated on ser bur not ser or ser as judged by immunoblotting using antibodies specific for the various phosphorylation sites. consistent with previous studies, stimulation of the cells with pma resulted in a modest increase in ser phosphorylation whilst forskolin resulted in increases in ser phosphorylation and, to a lesser extent, ser. co-expression of pim- and bad resulted in increased phosphorylation of ser and phosphorylation of ser and ser when compared to bad expressed in the absence of pim- similar results were also obtained for co-expression of pim- or pim- although pim- had a reduced effect on ser phosphorylation in bad compared to co-expression with pim- and pim-. the phosphorylation of bad by pim kinases was not further increased by pma stimulation, although ser phosphorylation was further increased by forskolin stimulation. the phosphorylation of bad was dependent on the kinase activity of pim kinases, as co-expression of kinase dead versions of pim- or and bad did not result in the phosphorylation of bad. the ability of pim- to phosphorylate bad in which one of the phosphorylation sites had been mutated was also determined by co-expression. as expected the mutation of ser to alanine prevented phosphorylation of this site by pim- but did not significantly affect the phosphorylation of either ser or ser. similarly, mutation of ser prevented phosphorylation of this site by pim- but did not significantly affect phosphorylation of ser surprisingly mutation of ser to alanine did cause a reduction in the level of ser phosphorylation by pim- and a similar reduction in ser phosphorylation was also seen in ser112/ser155ala mutants. mutation of ser to alanine not only blocked phosphorylation of ser by pim- but also greatly reduced ser and ser phosphorylation. mutation of ser to an acidic residue to mimic phosphorylation was able to restore phosphorylation of ser and ser. similar effects of the mutants were also seen in the absence of co-expressed pim- when cells were stimulated with agents to induce the phosphorylation of specific sites within bad. pma promotes bad ser phosphorylation, probably via rsk, however this was prevented by ser mutation. forskolin stimulated ser phosphorylation via pka, and this was also prevented by mutation of ser. pim promotes 14-3- binding to bad via phosphorylation of ser and dissociation from bcl-xl phosphorylation of a protein can create binding sites for 14-3- proteins, and ser and in bad lie in a potential consensus 14-3- motif. ser does not lie in a consensus 14-3- binding motif, however in vitro phosphorylation of bad by pka does promote 14-3- interaction. to test the ability of bad to interact with 14-3- gst-bad was co-expressed in hek- cells with pim- and 14-3- overlay assays were used to determine if bad could bind 14-3- proteins. in the overlay assay, recombinant 14-3- protein was used to detect the presence of proteins on a western blot capable of binding 14-3- gst-bad isolated by glutathione-sepharose pull downs from the cell lysates was tested for its ability to bind to 14-3- in the absence of pim- co-expression, gst-bad was not significantly phosphorylated on ser or and did not bind 14-3- in the overlay assay. co-expression of pim- however resulted in phosphorylation of all three sites and promoted interaction of the gst-bad with 14-3- in the overlay assay. mutation of either ser or ser to alanine resulted in a modest reduction in the ability of the pim- phosphorylated gst-bad to interact with 14-3- proteins in the overlay assay. as expected, mutation of ser to alanine, which also blocks the phosphorylation of ser and ser completely blocked the ability of gst-bad to interact with 14-3- proteins. mutation of ser to an acidic residue also blocked the ability of bad to bind to 14-3- proteins in the overlay assay, even though pim- was able to phosphorylate both ser and ser in the ser136asp or ser136glu bad mutants. these data suggest that ser is the major 14-3- binding site. consistent with this, a double mutant of both ser and ser to alanine, which was phosphorylated on ser, was still able to interact with 14-3- proteins in the overlay assay. similar to pim- when pim- or pim- were co-expressed with gst-bad, they were also able to promote 14-3- binding in the overlay assay. to test if pim kinases promoted the interaction of bad and 14-3- proteins in cells, pim kinases and gst-bad were co-expressed in hek- cells and the gst-bad isolated by glutathione-sepharose pull downs and blotted for the presence of endogenous 14-3- proteins using a pan 14-3- isoform antibody. in the absence of pim kinase co-expression, 14-3- proteins were not detected in the gst-bad pull downs, however expression of pim- or was found to promote the binding of 14-3- to gst-bad in cells. the ability of pim kinases to promote the interaction of bad and 14-3- was dependent on their kinase activity, as co-expression of kinase dead pim- or did not promote 14-3- binding as judged by either co-precipitation or 14-3- overlay assays. phosphorylation of bad has been shown to prevent an interaction with bcl-xl. the ability of pim kinases to prevent the interaction of gst-bad with endogenous bcl-xl was tested in pull down experiments. hela cells were used for these experiments as these cells express detectable levels of endogenous bcl-xl. as expected, gst-bad, which was not significantly phosphorylated in the absence of kinase co-expression, was able to interact with bcl-xl in hela cells. co-expression of pim- or resulted in phosphorylation of gst-bad and reduced the interaction of gst-bad with bcl-xl. this was dependent on the kinase activity of pim, as co-expression of kinase dead pim mutants did not have this effect. similar to the 14-3- interaction, ser appeared to be the critical residue involved in this process. mutation of ser ser, or both ser and ser to alanine in bad did not affect the ability of pim- to prevent the interaction of gst-bad and bcl-xl. mutation of ser in bad to alanine or an acidic residue blocked the ability of pim- to promote the dissociation of gst-bad and bcl-xl. discussion pim- or were found to be able to phosphorylate bad in vitro and in cells. pim- was found to be the most specific of the pim kinases for ser of bad in vitro, although it was also able to phosphorylate ser both in vitro and when over-expressed in hek- cells. pim- was a poor ser kinase in vitro and, consistent with this, was the least effective of all the pim kinases at phosphorylating this site in cells. in agreement with this pim- has previously been described to phosphorylate ser. interestingly, the phosphorylation of ser and ser by over-expression of pim- has been suggested to be dependent on the cell line used. both previous results and our results suggest this may reflect different over-expression levels of pim- achieved in the different cell types used. pim- was able to phosphorylate ser ser and ser in bad, however phosphorylation of ser was significantly more efficient than phosphorylation of the other sites in vitro. pim- has previously been found to phosphorylate ser in bad, although its ability to phosphorylate bad on other sites has not previously been reported. the ability of pim- to phosphorylate bad has not previously been addressed. here we report that pim- is also able to phosphorylate ser in bad. interestingly pim- was the most effective kinase in vitro for ser of bad. unlike pim- or pim- which demonstrated a marked preference for ser pim- was also effective at phosphorylating ser and to a lesser extent ser when bad protein was used as an in vitro substrate. in addition pim- was also able to phosphorylate other sites in bad including ser in vitro. this site has been reported to be phosphorylated in vivo, although the kinase responsible was not identified in the initial study. mutagenesis of ser in bad has suggested that phosphorylation of this site, like phosphorylation of ser and inhibits the pro-apoptotic activity of bad. phosphorylation of this site by pim- would therefore be consistent with the pro-survival function of pim- to the best of our knowledge, this study is the first to report a pim- cellular substrate. the interaction between the phosphorylation sites in bad and their roles in bad function is complex. phosphorylation of ser appears to be required for the phosphorylation of ser and ser to occur when bad is over-expressed in cells. similarly, ser appears to be the major 14-3- binding site in bad, while ser is suggested to be required for binding to bcl-xl. despite this, phosphorylation of endogenous bad on ser has proven hard to demonstrate in vivo. this may reflect the poor sensitivity of the antibodies available, however it has also been suggested that this site may not be physiologically important. in contrast to this, studies using transfected bad have suggested that phosphorylation of this site is essential for the phosphorylation of ser and ser and 14-3- binding, which are required to inhibit the apoptotic activity of bad. studies on ser have suggested that this site may be a'gatekeeper' site, and that dephosphorylation of this site is required to recruit a phosphatase that then dephosphorylates ser and ser. an extension of this idea lead to the proposal that pim- only directly phosphorylates ser and that an increase in ser phosphorylation when pim- was over-expressed was due to a decrease in the rate of ser dephosphorylation. whilst this cannot be completely excluded, it is not consistent with our data that pim- can promote the phosphorylation of a ser112ala bad mutant in cells. the interplay between the different bad phosphorylation sites is thus very complex and further work is required to completely understand these mechanisms. an added degree of complexity is the number of physiological kinases for bad, including pim-1- pkb, pak, rsk, pka and possibly other kinases potentially playing a role. the exact context in which pim phosphorylates bad in vivo may therefore depend on both cell type and stimulus and remains to be fully understood. the ability of pim kinases to phosphorylate bad is consistent with the suggested role of these kinases in pro survival or proliferation pathways. the finding that all three pim kinases can phosphorylate similar sites in bad could in part explain the redundancy between pim kinases in providing a cellular survival signal. the knockout of pim kinase might therefore be expected to have a severe phenotype, however a triple knockout of pim- and has recently been reported. surprisingly these mice were found to be viable, and while they were smaller in size than wild type mice they displayed no obvious health problems. these mice did however show reduced proliferation of some haematopoietic cells in response to growth factors. interestingly, a triple knockin mutation of bad in mice was also found to be viable and did not show increased levels of apoptosis in tissues under normal conditions. these mice also displayed reduced numbers of t and b cells and reduced cytokine induced survival of these cells in vitro, a phenotype with some similarity to that of the triple pim kinase knockout.  we present here biochemical evidence that all three isoforms of pim kinase are able to phosphorylate bad. the major site of bad phosphorylation for all three isoforms of pim kinase is ser however, we also demonstrate that pim kinases are able to phosphorylate bad to a lesser extent on ser and ser in vitro. interestingly pim- exhibits less specificity for ser than pim- or pim- and was the most effective pim isoform for ser and ser phosphorylation in bad in vitro. we also provide evidence that pim kinases may modulate the phosphorylation of bad on ser ser and ser in hek- cells. 
here we show that the pim kinases are constitutively active when expressed in hek- cells and are able to phosphorylate the bcl- family member bad on three residues, ser ser and ser in vitro and in cells. mutation of ser to alanine prevented the phosphorylation of ser and ser by pim kinases in hek- cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. this would be consistent with the proposed function of pim kinases in promoting cell proliferation and preventing cell death. over-expression of pim- and pim- in mice promotes the development of lymphomas, and up-regulation of pim expression has been observed in several human cancers.
 pim- and are a group of enzymes related to the calcium calmodulin family of protein kinases. over-expression of pim- and pim- in mice promotes the development of lymphomas, and up-regulation of pim expression has been observed in several human cancers.  here we show that the pim kinases are constitutively active when expressed in hek- cells and are able to phosphorylate the bcl- family member bad on three residues, ser ser and ser in vitro and in cells. in vitro mapping showed that pim- predominantly phosphorylated ser while pim- phosphorylated ser but also ser and ser at a reduced rate compared to ser pim- was found to be the least specific for ser and the most effective at phosphorylating ser and ser pim- was also able to phosphorylate other sites in bad in vitro, including ser another potential in vivo site. mutation of ser to alanine prevented the phosphorylation of ser and ser by pim kinases in hek- cells, suggesting that this site must be phosphorylated first in order to make the other sites accessible. pim phosphorylation of bad was also found to promote the 14-3- binding of bad and block its association with bcl-xl.  all three pim kinase family members predominantly phosphorylate bad on ser and in addition are capable of phosphorylating bad on multiple sites associated with the inhibition of the pro-apoptotic function of bad in hek- cells. this would be consistent with the proposed function of pim kinases in promoting cell proliferation and preventing cell death. 