BACKGROUND
membrane traffic to the furrow is an essential facet of cytokinesis  <cit> .
the plasma membrane of the furrow of dividing cells has a distinct lipid and protein composition as compared to the rest of the plasma membrane, with the furrow enriched in cholesterol and phosphatidylinositol  <dig>  5-bisphosphate  and various proteins involved in intracellular membrane trafficking   <cit> .
exocytosis of intracellular membranes occurs to the furrow and intercellular bridge, reflecting a requirement for the delivery of intracellular signalling and/or membrane re-modelling activities to the correct spatial co-ordinates during abscission  <cit> .
identifying the molecular basis of these trafficking events is crucial for a full understanding of cell division.
we previously have shown that rab <dig> and its effector molecule, rab11-fip <dig> , associate with recycling endosomes and traffic into the furrow and midbody  <cit> .
depletion of fip <dig> or expression of a fip <dig> mutant unable to bind rab <dig> results in defective abscission, and both these genes are over-expressed in cancer  <cit> .
fip <dig> can bind both rab <dig> and arf <dig> gtpases simultaneously, and fip <dig>  rab <dig> and arf <dig> form a ternary complex in vitro  <cit> , prompting us to suggest that multiple interactions between rab <dig> and arf <dig> with the exocyst complex may serve to anchor fip3-containing vesicles in the midbody prior to abscission  <cit> , where they function as an organisation platform for the assembly of the abscission machinery  <cit> .
recent studies have also suggested that a dynamic interaction of fip <dig> with the centralspindlin component cyk- <dig> may also contribute to the localisation of fip <dig> during mitosis  <cit> .
fip <dig> exhibits profound spatial and temporal dynamics during cell division  <cit> .
gfp-fip <dig> redistributes from diffuse cytosolic staining onto membranes at the centrosome during early anaphase; gfp-fip <dig> then begins to accumulate in the developing furrow before entering the midbody and at very late telophase accumulating at or close to the midbody ring.
similar distributions have recently been reported for endogenous fip <dig>  <cit> .
in addition to its role in mitosis, fip <dig> has also been suggested to control the spatial distribution of recycling endosomes in interphase cells  <cit> , raising the question of how these distinct functions are modulated.
recent work has revealed that fip <dig> is a phospho-protein with multiple phospho-acceptor sites  <cit> .
phosphorylation of residues s- <dig>  s- <dig>  s- <dig> and s- <dig> modulates the interaction of fip <dig> with motor machinery, thereby controlling the spatial distribution of endosomes  <cit> .
however, there is no published work relating phosphorylation of fip <dig> at these or other sites to events in the cell cycle.
here, we identify and characterise four sites of fip <dig> phosphorylation: s- <dig>  s- <dig>  s- <dig> and s- <dig>  we show that cdk <dig>  which is active in metaphase  <cit> , phosphorylates fip <dig> exclusively on s- <dig> in vitro, and that s- <dig> phosphorylation of fip <dig> declines in parallel with cyclin b levels as cells exit metaphase and proceed into telophase.
a phospho-mimetic mutation of this site  exerted a modest effect on cytokinesis.
individual mutants at s- <dig>  s- <dig> and s- <dig> or a mutant of fip <dig> in which s- <dig>  s- <dig>  s- <dig> and s- <dig> were all mutated to alanine had no effect on cytokinesis and did not modulate fip <dig> distribution during the cell cycle.
we further show that fip <dig> is a cell cycle-regulated phosphoprotein and show that the shift in fip <dig> distribution from membranes to cytosol occurs concomitantly with dephosphorylation of fip <dig>  but that dephosphorylation of fip <dig> alone is insufficient to promote membrane association in vitro.
of the sites identified here, we show that s <dig> is dephosphorylated as cells move from metaphase into telophase.
finally, we show that phospho-mimetic or phospho-resistant mutants of s- <dig>  s- <dig>  s- <dig> or s- <dig> do not modulate transferrin uptake or recycling endosome distribution in interphase cells.
RESULTS
gfp-tagged fip <dig> undergoes a re-distribution from a diffuse cytosolic distribution to membrane compartment centred over the centrosome as cells progress from metaphase to anaphase  <cit> .
during early telophase, gfp-fip <dig> accumulates near dna but at the opposite side of the centrosomes, before gradually moving to mid-zone microtubules and then eventually translocating into the midbody in late telophase, just prior to abscission  <cit> .
a similar pattern has recently been reported for endogenous fip <dig>  <cit> .
we performed a simple subcellular fractionation of hela cell homogenates isolated at prophase/metaphase or telophase and studied the distribution of the endogenous protein between membrane and cytosolic fractions .
fip <dig> was found to re-distribute from the cytosol to membrane fractions in telophase, consistent with previous imaging studies  <cit> .
previous studies have suggested that fip <dig> is a phosphoprotein, with over  <dig> sites of phosphorylation identified  <cit> .
we hypothesised that phosphorylation/dephosphorylation may represent a potential mechanism for this regulation of fip <dig> localisation or function.
we immunoprecipitated fip <dig> from hela cells arrested in metaphase, and examined the distribution of fip <dig> on 2d-gel electrophoresis by immunoblotting and compared the signal to that obtained from identical samples treated with alkaline phosphatase .
phosphatase treatment consistently reduced the number of fip3-immunoreactive species identified, consistent with the notion that fip <dig> is phosphorylated in metaphase.
we identified four phospho-peptides by mass spectrometry , which encompassed s <dig>  s <dig>  s <dig> and s <dig> .
although not all of these sites were observed in the previous study  <cit> , it should be noted that we have focussed our study on fip <dig> in metaphase.
fip <dig> is phosphorylated by cdk <dig> in vitro on s102
we next generated a phospho-selective antibody to detect phosphorylation of residue s <dig>  this reagent readily detected recombinant fip <dig> purified from baculovirus, and also recombinant fip <dig> phosphorylated on s <dig> by incubation with cdk1-cyclin b ; the signal was selectively blocked by the phosphorylated antigenic peptide, but not the corresponding non-phosphorylated control peptide .
this antibody also readily detected phosphorylation of s <dig> within the context of gfp-fip <dig> ectopically expressed in hela cells  .
we found that total levels of phosphorylation of s <dig> within gfp-fip <dig> decreased as cells progressed from metaphase  to telophase  but total cellular levels of gfp-fip <dig>  were unchanged .
phospho-fip <dig> could be detected only within the cytosolic pool of fip <dig>  and levels of phosphorylation declined markedly as cells progressed from metaphase to telophase .
s <dig> is within in a consensus sequence similar to that for proline-directed kinases, such as cyclin-dependent kinase  <dig> .
consistent with this, figure  <dig> shows that recombinant cdk1-cyclin b readily phosphorylates baculovirus expressed recombinant fip <dig>  mass spectrometry revealed that cdk <dig> had phosphorylated recombinant fip <dig> only on residue s <dig> .
we also performed in vitro kinase assays with plk <dig>  aurora a and aurora b; no specific phosphorylation events could be identified .
attempts to generate phospho-specific antibodies to the other sites identified in figure  <dig> have thus far proven unsuccessful.
hence, we cannot ascertain whether phosphorylation of s <dig>  s <dig> or s <dig> is cell cycle-dependent .
consequences of fip <dig> phosphorylation
in an attempt to determine the role of phosphorylation of fip <dig> at these sites, we generated a panel of either s > a or s > d mutants within the context of gfp-fip <dig>  expression of these mutants in hela cells  did not affect cell growth .
we next examined whether over-expression of these mutants gave rise to defective cytokinesis by counting binuclear cells.
a modest increase in the fraction of binuclear cells was observed in cells expressing gfp-fip3-s102d.
however, this effect was smaller than was observed in cells expressing gfp-fip3-i737e, a rab11-binding deficient mutant of fip <dig>  previously shown to increase the number of binuclear cells  <cit>  despite similar levels of expression of these mutants .
similarly, the membrane/cytosol distribution of gfp-fip3-s102d revealed a modest decrease in ratio in metaphase compared to gfp-fip3-s102a or gfp-fip <dig> .
such data support the possibility that de/phosphorylation of s <dig> is involved in cell division.
surprisingly, when expressed in hela cells as gfp-fusion proteins, the distribution of the individual s > a or s > d mutants of fip <dig> appeared indistinguishable from wild-type fip <dig> throughout the stages of the cell cycle.
figure 5d shows the localisation in early and late telophase for gfp-s102a and gfp-s102d.
similar results were observed when all other mutants were expressed .
phosphorylation of ser- <dig>   <dig>   <dig> and  <dig> is not required for cytokinesis
recent studies have identified more than  <dig> phospho-acceptor sites on fip <dig> and its fly homologue nuclear fallout   <cit> .
mutation of four phospho-acceptor sites within fip <dig> to alanine  was also shown to modulate recycling endosome directionality, perhaps by regulating the interaction of fip <dig> with dynein  <cit> .
since we and others have proposed that the accumulation of fip3-containing vesicles in the midbody is a crucial facet of cytokinesis  <cit> , we posited that the de/phosphorylation of fip <dig> at these sites may control the interaction of fip3-positive vesicles with the microtubule network and thus the distribution of fip <dig> in the cell cycle.
however, over-expression of ha-fip <dig>  and ha-fip3- revealed that the 4a mutant did not affect cytokinesis, as revealed by quantification of the levels of binucleate cells .
the membrane/cytosol ratio of ha-fip <dig>  and ha-fip3- in metaphase were similar , consistent with these mutations not exerting a significant effect on the interaction of fip <dig> with membranes in metaphase.
similarly, the cell cycle distribution of ha-fip3-4a was indistinguishable from wild-type ha-fip <dig> .
it will be necessary to examine the distribution of the corresponding aspartate mutants to definitely rule out a role for phosphorylation/dephosphorylation of these residues, but the alanine mutant employed here strongly suggests that phosphorylation/dephosphorylation of these sites may not play a crucial role in cytokinesis.
phosphorylation of fip <dig> does not modulate gfp-fip <dig> distribution or recycling endosome function in interphase cells
the functional role of fip <dig> is not confined to events in the cell cycle, but studies have revealed an important role for fip <dig> in the control of recycling endosome distribution and function  <cit> .
previous studies have suggested that expression of gfp-fip <dig> results in a clustering of fip <dig> into a peri-nuclear region, concomitant with a clustering of recycling endosomes  <cit> .
expression of these mutant fip <dig> species reveal that this clustering is unaffected by either the phospho-mimetic  or phospho-resistant mutations .
this is in marked contrast to expression of gfp-fip3-i737e, a rab11-binding deficient mutant, which is predominantly cytosolic, consistent with a role for rab <dig> in the recruitment of fip <dig> to membranes  <cit> .
consistent with this, analysis of the uptake of fluorescently labelled transferrin revealed significant accumulation of label into the clustered gfp-fip <dig> compartment, and that the corresponding s > d  and s > a  mutants were without effect on transferrin accumulation.
the data shown in figure  <dig> are from cells exposed to labelled transferrin for  <dig> minutes; analysis of earlier time points  revealed no difference in accumulation between wild-type and phospho-mutants of fip <dig> .
thus, the phospho-acceptor sites identified here do not appear to modulate either the clustering of recycling endosomes induced by fip <dig> over-expression, or the ability of cargo to traffic into these endosomes.
similar data has been reported for the ha-fip3- mutant  <cit> .
fip <dig> is a cell cycle-regulated phospho-protein
the data presented above suggest that the phospho-acceptor sites identified here  play at best a minimal role in the control of fip <dig> distribution of function.
one explanation for this is that perhaps other, as yet unstudied, sites may be either more important or act cooperatively with those described here.
returning to our initial hypothesis that phosphorylation may represent a potential mechanism for the regulation of fip <dig> cytosol/membrane ratio in the cell cycle, we used a phospho-protein affinity column to selectively bind phosphorylated proteins from hela extracts, isolated from prophase/metaphase arrested cells or from cells in telophase .
the phospho-affinity column retained fip <dig> from prophase/metaphase arrested cells, but the extent of fip <dig> binding was considerably reduced in telophase extracts.
such data suggest that fip <dig> is phosphorylated in early stages of the cell cycle  and dephosphorylated in later stages .
as this corresponds to a movement of fip <dig> from largely cytosolic  to membrane associated , we tested this hypothesis by examining the membrane/cytosol distribution of fip <dig> in metaphase homogenates, treated with or without phosphatase, and then subsequently separated into membrane and cytosol fractions.
as revealed in figure 7b, dephosphorylation of fip <dig> consistently resulted in more fip <dig> distributing to the membrane fraction.
such data support the hypothesis that fip <dig> is a phosphoprotein.
as a control for these experiments, we examined the distribution of another rab <dig> effector, rab coupling protein /fip <dig>  the distribution of rcp/fip <dig> between membrane and cytosolic fractions was not altered during the cell cycle .
similarly, the levels of phosphorylated rcp/fip <dig> did not differ between metaphase and telophase .
such data suggest that the cell cycle-induced changes in localization and phosphorylation are specific to fip <dig>  although a modest change in membrane/cytosol ratio was observed for over-expressed gfp-fip3-s102d, this did not approach the magnitude reported here for gfp-fip <dig> .
nonetheless, these data strongly support the notion that the cytosol to membrane translocation of fip <dig> occurs concomitantly with dephosphorylation.
in an attempt to determine whether this was a direct effect, we incubated recombinant fip <dig>  with either artificial lipid vesicles or membranes from hela cells.
we could not detect any direct interaction under these conditions .
discussion
fip <dig> is a rab <dig> binding protein essential for the completion of abscission  <cit> .
previous work from our group and others has shown that fip <dig> exhibits considerable changes in subcellular distribution throughout the cell cycle.
here, we have investigated the potential role of phosphorylation as a mechanism to control fip <dig> distribution.
we have shown that fip <dig> is a cell cycle-regulated phosphoprotein , and that levels of phosphorylation of fip <dig> decrease as cells move from anaphase into telophase .
this dephosphorylation of fip <dig> is accompanied by a shift of fip <dig> from a largely cytosolic fraction to a membrane fraction , consistent with the observed distribution of gfp-fip <dig> reported by us and others  <cit> .
using ms approaches we have identified four putative sites of fip <dig> phosphorylation: s <dig>  s <dig>  s <dig> and s <dig> , extending the list of potential phospho-acceptor sites within fip <dig> - see figure 2c <cit> .
of these, we show that phosphorylation of s <dig> is cell cycle regulated.
s <dig> lies within a consensus site for proline-directed kinases, including cdk <dig>  <cit> .
since cdk <dig> activity is tightly regulated during the cell cycle  <cit> , we tested the hypothesis that cdk1-cyclin b could phosphorylate this site in vitro.
figure  <dig> shows that recombinant cdk1-cyclin b phosphorylated recombinant fip <dig> in vitro solely on s <dig>  consistent with cdk1-cyclin b kinase phosphorylating fip <dig> in vivo, levels of ps102-fip <dig> fall as cells progress from anaphase into telophase .
in an attempt to determine the functional significance of this phospho-acceptor site, we generated s102a and s102d mutants and expressed these in hela cells.
expression of gfp-fip3-s102d but not gfp-fip3-s102a resulted in a modest but reproducible increase in the number of binucleate cells, consistent with a defect in cytokinesis .
however, this effect was significantly less than that observed upon expression of gfp-fip3-i737e, a rab11-binding defective mutant  <cit> .
the molecular basis of the role of s- <dig> in cytokinesis remains to be established .
we examined the functional consequences of s > a or s > d mutation at all of the sites identified here .
with the exception of s102d, over-expression of these mutants in hela cells had no effect on the numbers of binucleate cells or the distribution of fip <dig> in the cell cycle .
of note, we also expressed all combinations of double, triple and quadruple aspartate mutants.
none of these exhibited significant increases in the numbers of binucleate cells .
similarly, the interphase role of fip <dig> in the spatial control of the recycling endosomal compartment and in transferrin recycling did not appear to be impaired by over-expression of any of the phospho-mutants .
one exception to this concerns gfp-fip3-s450a.
we consistently observed that transfection of hela cells with gfp-fip3-s450a led to significant cell death, with little, if any, immuno-reactive gfp-fip3-s450a observed .
by contrast, gfp-fip3-s450d did not modulate cell growth, and its localisation was identical to that of gfp-fip <dig>  interestingly, when double, triple or quadruple phospho-mutants were generated, any mutant containing s450a failed to express .
such observations suggest that this residue may be involved in fip <dig> stability, processing or folding, but further studies will be required to determine the basis of this.
more than  <dig> phospho-acceptor sites on fip <dig>  have recently been identified  <cit> .
phosphorylation of nuf by ikk-ε has been shown to regulate the trafficking of rab11-positive recycling endosomal-derived vesicles into and out of the fly bristle tip by coordinating its interaction with dynein  <cit> .
mutation of s- <dig>   <dig>   <dig> and  <dig> to a  was also shown to modulate the ability of ikk-ε to control recycling endosome directionality  <cit> .
we hypothesised that de/phosphorylation of fip <dig> at these sites may control the interaction of fip3-positive vesicles with the microtubule network and thus modulate the function and/or distribution of fip <dig> in the cell cycle.
however, over-expression of ha-fip <dig>  or ha-fip3- did not affect cytokinesis as revealed by quantification of the levels of binucleate cells .
similarly, the membrane/cytosol ratio of ha-fip <dig>  and ha-fip3- in metaphase were similar , consistent with these mutations not exerting a significant effect on the interaction of fip <dig> with membranes.
similarly, the distribution of this mutant during the cell cycle was indistinguishable from the ha-tagged wild-type fip <dig> .
fip <dig> is a cell cycle regulated phosphoprotein
the identification of a range of phospho-acceptor sites within fip <dig> begs the question of the functional role of these sites.
given the significant redistribution of gfp-tagged fip <dig> from cytosol to membranes  during the cell cycle and our observation that s- <dig> is dephosphorylated during progression from metaphase to telophase , we further explored whether phosphorylation of fip <dig> is modulated during the cell cycle.
to test this, we used a phospho-protein affinity column to selectively bind phosphorylated proteins from hela extracts, isolated from prophase/metaphase arrested cells or from cells in telophase .
the phospho-affinity column retained fip <dig> from prophase/metaphase arrested cells, but the extent of fip <dig> binding was considerably reduced in telophase extracts.
such data suggest that fip <dig> is phosphorylated in early stages of the cell cycle  and dephosphorylated in later stages .
as this corresponds to a movement of fip <dig> from largely cytosolic  to membrane associated , we tested this hypothesis by examining the membrane/cytosol distribution of fip <dig> in metaphase homogenates, treated with or without phosphatase, and then subsequently separated into membrane and cytosol fractions.
as revealed in figure 7b, dephosphorylation of fip <dig> consistently resulted in more fip <dig> distributing to the membrane fraction.
such data support the hypothesis that fip <dig> is a phosphoprotein.
as a control for these experiments, we examined the distribution of another rab <dig> effector, rab coupling protein /fip <dig>  the distribution of rcp/fip <dig> between membrane and cytosolic fractions was not altered during the cell cycle.
similarly, the levels of phosphorylated rcp/fip <dig> did not differ between metaphase and telophase.
such data suggest that the cell cycle-induced changes in localization and phosphorylation are specific to fip <dig>
these data suggest that dephosphorylation of fip <dig> accompanies its redistribution from a cytosolic fraction to a membrane fraction.
several lines of argument suggest that dephosphorylation of fip <dig> alone is unlikely to be sufficient to mediate this.
firstly, we and others have shown that fip <dig> is recruited to membranes via interaction with rab <dig>  <cit> .
indeed, inspection of the data in figure  <dig> reveals that a fip <dig> mutant  unable to bind rab <dig> is cytosolic  <cit> .
hence, the interaction with rab <dig> is likely to be the most important factor controlling fip <dig> membrane distribution.
secondly, recombinant fip <dig> does not bind either to phospholipid vesicles or to intracellular membranes isolated from hela cells .
finally, neither s > d nor s > a mutants exhibited altered distribution of gfp-fip <dig> throughout the cell cycle .
collectively, these data suggest that although fip <dig> is a cell cycle-regulated phosphoprotein, the consequences of phosphorylation are not primarily to control the localisation of fip <dig>  this is also consistent with the location of the phospho-sites within fip3: all four sites studied here are located towards the n-terminal domain of fip <dig>  and are not within the coiled-coil domain which encompasses the sites of rab <dig>  arf <dig> and cyk- <dig> interaction   <cit> .
it should be noted that fip <dig> has multiple other potential phospho-acceptor sites, and over  <dig> such sites have been reported to be phosphorylated in vitro  <cit> .
some of these sites are located within the coiled-coil domain, and may modulate the interaction of fip <dig> with other proteins or possibly control fip <dig> homo-dimerisation.
mutation of the sites reported here did not in our hands modulate the distribution of dynein , nor affect the re-distribution of recycling endosomes characteristic of fip <dig> over-expression .
similarly, traffic of cargo into and out of the clustered endosomes was not modulated by either s > a or s > d mutation of these sites singly  or collectively.
CONCLUSIONS
here we have shown that fip <dig> is a cell cycle regulated phosphoprotein, and that fip <dig> dephosphorylation occurs concomitantly with the shift from largely cytosolic to membrane bound observed as cells pass from metaphase to telophase.
the phospho-acceptor sites studied here do not appear to control these changes.
there are several possible explanations for this.
first, the sites identified are not involved in this interaction, or that multiple sites of de/phosphorylation are involved.
alternatively, it is possible that multiple mechanisms of fip <dig> targeting exist: this is consistent with studies showing that rab <dig>  arf <dig> and ect <dig> are all binding partners for fip <dig>  and all may contribute to fip <dig> localisation.
it is unlikely that a single site of phosphorylation may control all of these interactions.
we favour a model in which the membrane association of fip <dig> is driven mainly by interaction with rab <dig>  and that dephosphorylation of fip <dig> may represent a distinct regulatory mechanism, perhaps involving motor protein interactions resulting in the accumulation of fip <dig> in midbodies.
further work will be required to resolve these models.

 membrane traffic to the furrow is an essential facet of cytokinesis. the plasma membrane of the furrow of dividing cells has a distinct lipid and protein composition as compared to the rest of the plasma membrane, with the furrow enriched in cholesterol and phosphatidylinositol 5-bisphosphate and various proteins involved in intracellular membrane trafficking. exocytosis of intracellular membranes occurs to the furrow and intercellular bridge, reflecting a requirement for the delivery of intracellular signalling and/or membrane re-modelling activities to the correct spatial co-ordinates during abscission. identifying the molecular basis of these trafficking events is crucial for a full understanding of cell division. we previously have shown that rab and its effector molecule, rab11-fip, associate with recycling endosomes and traffic into the furrow and midbody. depletion of fip or expression of a fip mutant unable to bind rab results in defective abscission, and both these genes are over-expressed in cancer. fip can bind both rab and arf gtpases simultaneously, and fip rab and arf form a ternary complex in vitro, prompting us to suggest that multiple interactions between rab and arf with the exocyst complex may serve to anchor fip3-containing vesicles in the midbody prior to abscission, where they function as an organisation platform for the assembly of the abscission machinery. recent studies have also suggested that a dynamic interaction of fip with the centralspindlin component cyk- may also contribute to the localisation of fip during mitosis. fip exhibits profound spatial and temporal dynamics during cell division. gfp-fip redistributes from diffuse cytosolic staining onto membranes at the centrosome during early anaphase; gfp-fip then begins to accumulate in the developing furrow before entering the midbody and at very late telophase accumulating at or close to the midbody ring. similar distributions have recently been reported for endogenous fip. in addition to its role in mitosis, fip has also been suggested to control the spatial distribution of recycling endosomes in interphase cells, raising the question of how these distinct functions are modulated. recent work has revealed that fip is a phospho-protein with multiple phospho-acceptor sites. phosphorylation of residues s- s- s- and s- modulates the interaction of fip with motor machinery, thereby controlling the spatial distribution of endosomes. however, there is no published work relating phosphorylation of fip at these or other sites to events in the cell cycle. here, we identify and characterise four sites of fip phosphorylation: s- s- s- and s- we show that cdk which is active in metaphase, phosphorylates fip exclusively on s- in vitro, and that s- phosphorylation of fip declines in parallel with cyclin b levels as cells exit metaphase and proceed into telophase. a phospho-mimetic mutation of this site exerted a modest effect on cytokinesis. individual mutants at s- s- and s- or a mutant of fip in which s- s- s- and s- were all mutated to alanine had no effect on cytokinesis and did not modulate fip distribution during the cell cycle. we further show that fip is a cell cycle-regulated phosphoprotein and show that the shift in fip distribution from membranes to cytosol occurs concomitantly with dephosphorylation of fip but that dephosphorylation of fip alone is insufficient to promote membrane association in vitro. of the sites identified here, we show that s is dephosphorylated as cells move from metaphase into telophase. finally, we show that phospho-mimetic or phospho-resistant mutants of s- s- s- or s- do not modulate transferrin uptake or recycling endosome distribution in interphase cells.  gfp-tagged fip undergoes a re-distribution from a diffuse cytosolic distribution to membrane compartment centred over the centrosome as cells progress from metaphase to anaphase. during early telophase, gfp-fip accumulates near dna but at the opposite side of the centrosomes, before gradually moving to mid-zone microtubules and then eventually translocating into the midbody in late telophase, just prior to abscission. a similar pattern has recently been reported for endogenous fip. we performed a simple subcellular fractionation of hela cell homogenates isolated at prophase/metaphase or telophase and studied the distribution of the endogenous protein between membrane and cytosolic fractions. fip was found to re-distribute from the cytosol to membrane fractions in telophase, consistent with previous imaging studies. previous studies have suggested that fip is a phosphoprotein, with over sites of phosphorylation identified. we hypothesised that phosphorylation/dephosphorylation may represent a potential mechanism for this regulation of fip localisation or function. we immunoprecipitated fip from hela cells arrested in metaphase, and examined the distribution of fip on 2d-gel electrophoresis by immunoblotting and compared the signal to that obtained from identical samples treated with alkaline phosphatase. phosphatase treatment consistently reduced the number of fip3-immunoreactive species identified, consistent with the notion that fip is phosphorylated in metaphase. we identified four phospho-peptides by mass spectrometry, which encompassed s s s and s. although not all of these sites were observed in the previous study, it should be noted that we have focussed our study on fip in metaphase. fip is phosphorylated by cdk in vitro on s102 we next generated a phospho-selective antibody to detect phosphorylation of residue s this reagent readily detected recombinant fip purified from baculovirus, and also recombinant fip phosphorylated on s by incubation with cdk1-cyclin b; the signal was selectively blocked by the phosphorylated antigenic peptide, but not the corresponding non-phosphorylated control peptide. this antibody also readily detected phosphorylation of s within the context of gfp-fip ectopically expressed in hela cells. we found that total levels of phosphorylation of s within gfp-fip decreased as cells progressed from metaphase to telophase but total cellular levels of gfp-fip were unchanged. phospho-fip could be detected only within the cytosolic pool of fip and levels of phosphorylation declined markedly as cells progressed from metaphase to telophase. s is within in a consensus sequence similar to that for proline-directed kinases, such as cyclin-dependent kinase. consistent with this, figure shows that recombinant cdk1-cyclin b readily phosphorylates baculovirus expressed recombinant fip mass spectrometry revealed that cdk had phosphorylated recombinant fip only on residue s. we also performed in vitro kinase assays with plk aurora a and aurora b; no specific phosphorylation events could be identified. attempts to generate phospho-specific antibodies to the other sites identified in figure have thus far proven unsuccessful. hence, we cannot ascertain whether phosphorylation of s s or s is cell cycle-dependent. consequences of fip phosphorylation in an attempt to determine the role of phosphorylation of fip at these sites, we generated a panel of either s > a or s > d mutants within the context of gfp-fip expression of these mutants in hela cells did not affect cell growth. we next examined whether over-expression of these mutants gave rise to defective cytokinesis by counting binuclear cells. a modest increase in the fraction of binuclear cells was observed in cells expressing gfp-fip3-s102d. however, this effect was smaller than was observed in cells expressing gfp-fip3-i737e, a rab11-binding deficient mutant of fip previously shown to increase the number of binuclear cells despite similar levels of expression of these mutants. similarly, the membrane/cytosol distribution of gfp-fip3-s102d revealed a modest decrease in ratio in metaphase compared to gfp-fip3-s102a or gfp-fip. such data support the possibility that de/phosphorylation of s is involved in cell division. surprisingly, when expressed in hela cells as gfp-fusion proteins, the distribution of the individual s > a or s > d mutants of fip appeared indistinguishable from wild-type fip throughout the stages of the cell cycle. figure 5d shows the localisation in early and late telophase for gfp-s102a and gfp-s102d. similar results were observed when all other mutants were expressed. phosphorylation of ser- and is not required for cytokinesis recent studies have identified more than phospho-acceptor sites on fip and its fly homologue nuclear fallout. mutation of four phospho-acceptor sites within fip to alanine was also shown to modulate recycling endosome directionality, perhaps by regulating the interaction of fip with dynein. since we and others have proposed that the accumulation of fip3-containing vesicles in the midbody is a crucial facet of cytokinesis, we posited that the de/phosphorylation of fip at these sites may control the interaction of fip3-positive vesicles with the microtubule network and thus the distribution of fip in the cell cycle. however, over-expression of ha-fip and ha-fip3- revealed that the 4a mutant did not affect cytokinesis, as revealed by quantification of the levels of binucleate cells. the membrane/cytosol ratio of ha-fip and ha-fip3- in metaphase were similar, consistent with these mutations not exerting a significant effect on the interaction of fip with membranes in metaphase. similarly, the cell cycle distribution of ha-fip3-4a was indistinguishable from wild-type ha-fip. it will be necessary to examine the distribution of the corresponding aspartate mutants to definitely rule out a role for phosphorylation/dephosphorylation of these residues, but the alanine mutant employed here strongly suggests that phosphorylation/dephosphorylation of these sites may not play a crucial role in cytokinesis. phosphorylation of fip does not modulate gfp-fip distribution or recycling endosome function in interphase cells the functional role of fip is not confined to events in the cell cycle, but studies have revealed an important role for fip in the control of recycling endosome distribution and function. previous studies have suggested that expression of gfp-fip results in a clustering of fip into a peri-nuclear region, concomitant with a clustering of recycling endosomes. expression of these mutant fip species reveal that this clustering is unaffected by either the phospho-mimetic or phospho-resistant mutations. this is in marked contrast to expression of gfp-fip3-i737e, a rab11-binding deficient mutant, which is predominantly cytosolic, consistent with a role for rab in the recruitment of fip to membranes. consistent with this, analysis of the uptake of fluorescently labelled transferrin revealed significant accumulation of label into the clustered gfp-fip compartment, and that the corresponding s > d and s > a mutants were without effect on transferrin accumulation. the data shown in figure are from cells exposed to labelled transferrin for minutes; analysis of earlier time points revealed no difference in accumulation between wild-type and phospho-mutants of fip. thus, the phospho-acceptor sites identified here do not appear to modulate either the clustering of recycling endosomes induced by fip over-expression, or the ability of cargo to traffic into these endosomes. similar data has been reported for the ha-fip3- mutant. fip is a cell cycle-regulated phospho-protein the data presented above suggest that the phospho-acceptor sites identified here play at best a minimal role in the control of fip distribution of function. one explanation for this is that perhaps other, as yet unstudied, sites may be either more important or act cooperatively with those described here. returning to our initial hypothesis that phosphorylation may represent a potential mechanism for the regulation of fip cytosol/membrane ratio in the cell cycle, we used a phospho-protein affinity column to selectively bind phosphorylated proteins from hela extracts, isolated from prophase/metaphase arrested cells or from cells in telophase. the phospho-affinity column retained fip from prophase/metaphase arrested cells, but the extent of fip binding was considerably reduced in telophase extracts. such data suggest that fip is phosphorylated in early stages of the cell cycle and dephosphorylated in later stages. as this corresponds to a movement of fip from largely cytosolic to membrane associated, we tested this hypothesis by examining the membrane/cytosol distribution of fip in metaphase homogenates, treated with or without phosphatase, and then subsequently separated into membrane and cytosol fractions. as revealed in figure 7b, dephosphorylation of fip consistently resulted in more fip distributing to the membrane fraction. such data support the hypothesis that fip is a phosphoprotein. as a control for these experiments, we examined the distribution of another rab effector, rab coupling protein /fip the distribution of rcp/fip between membrane and cytosolic fractions was not altered during the cell cycle. similarly, the levels of phosphorylated rcp/fip did not differ between metaphase and telophase. such data suggest that the cell cycle-induced changes in localization and phosphorylation are specific to fip although a modest change in membrane/cytosol ratio was observed for over-expressed gfp-fip3-s102d, this did not approach the magnitude reported here for gfp-fip. nonetheless, these data strongly support the notion that the cytosol to membrane translocation of fip occurs concomitantly with dephosphorylation. in an attempt to determine whether this was a direct effect, we incubated recombinant fip with either artificial lipid vesicles or membranes from hela cells. we could not detect any direct interaction under these conditions. discussion fip is a rab binding protein essential for the completion of abscission. previous work from our group and others has shown that fip exhibits considerable changes in subcellular distribution throughout the cell cycle. here, we have investigated the potential role of phosphorylation as a mechanism to control fip distribution. we have shown that fip is a cell cycle-regulated phosphoprotein, and that levels of phosphorylation of fip decrease as cells move from anaphase into telophase. this dephosphorylation of fip is accompanied by a shift of fip from a largely cytosolic fraction to a membrane fraction, consistent with the observed distribution of gfp-fip reported by us and others. using ms approaches we have identified four putative sites of fip phosphorylation: s s s and s, extending the list of potential phospho-acceptor sites within fip - see figure 2c. of these, we show that phosphorylation of s is cell cycle regulated. s lies within a consensus site for proline-directed kinases, including cdk. since cdk activity is tightly regulated during the cell cycle, we tested the hypothesis that cdk1-cyclin b could phosphorylate this site in vitro. figure shows that recombinant cdk1-cyclin b phosphorylated recombinant fip in vitro solely on s consistent with cdk1-cyclin b kinase phosphorylating fip in vivo, levels of ps102-fip fall as cells progress from anaphase into telophase. in an attempt to determine the functional significance of this phospho-acceptor site, we generated s102a and s102d mutants and expressed these in hela cells. expression of gfp-fip3-s102d but not gfp-fip3-s102a resulted in a modest but reproducible increase in the number of binucleate cells, consistent with a defect in cytokinesis. however, this effect was significantly less than that observed upon expression of gfp-fip3-i737e, a rab11-binding defective mutant. the molecular basis of the role of s- in cytokinesis remains to be established. we examined the functional consequences of s > a or s > d mutation at all of the sites identified here. with the exception of s102d, over-expression of these mutants in hela cells had no effect on the numbers of binucleate cells or the distribution of fip in the cell cycle. of note, we also expressed all combinations of double, triple and quadruple aspartate mutants. none of these exhibited significant increases in the numbers of binucleate cells. similarly, the interphase role of fip in the spatial control of the recycling endosomal compartment and in transferrin recycling did not appear to be impaired by over-expression of any of the phospho-mutants. one exception to this concerns gfp-fip3-s450a. we consistently observed that transfection of hela cells with gfp-fip3-s450a led to significant cell death, with little, if any, immuno-reactive gfp-fip3-s450a observed. by contrast, gfp-fip3-s450d did not modulate cell growth, and its localisation was identical to that of gfp-fip interestingly, when double, triple or quadruple phospho-mutants were generated, any mutant containing s450a failed to express. such observations suggest that this residue may be involved in fip stability, processing or folding, but further studies will be required to determine the basis of this. more than phospho-acceptor sites on fip have recently been identified. phosphorylation of nuf by ikk-ε has been shown to regulate the trafficking of rab11-positive recycling endosomal-derived vesicles into and out of the fly bristle tip by coordinating its interaction with dynein. mutation of s- and to a was also shown to modulate the ability of ikk-ε to control recycling endosome directionality. we hypothesised that de/phosphorylation of fip at these sites may control the interaction of fip3-positive vesicles with the microtubule network and thus modulate the function and/or distribution of fip in the cell cycle. however, over-expression of ha-fip or ha-fip3- did not affect cytokinesis as revealed by quantification of the levels of binucleate cells. similarly, the membrane/cytosol ratio of ha-fip and ha-fip3- in metaphase were similar, consistent with these mutations not exerting a significant effect on the interaction of fip with membranes. similarly, the distribution of this mutant during the cell cycle was indistinguishable from the ha-tagged wild-type fip. fip is a cell cycle regulated phosphoprotein the identification of a range of phospho-acceptor sites within fip begs the question of the functional role of these sites. given the significant redistribution of gfp-tagged fip from cytosol to membranes during the cell cycle and our observation that s- is dephosphorylated during progression from metaphase to telophase, we further explored whether phosphorylation of fip is modulated during the cell cycle. to test this, we used a phospho-protein affinity column to selectively bind phosphorylated proteins from hela extracts, isolated from prophase/metaphase arrested cells or from cells in telophase. the phospho-affinity column retained fip from prophase/metaphase arrested cells, but the extent of fip binding was considerably reduced in telophase extracts. such data suggest that fip is phosphorylated in early stages of the cell cycle and dephosphorylated in later stages. as this corresponds to a movement of fip from largely cytosolic to membrane associated, we tested this hypothesis by examining the membrane/cytosol distribution of fip in metaphase homogenates, treated with or without phosphatase, and then subsequently separated into membrane and cytosol fractions. as revealed in figure 7b, dephosphorylation of fip consistently resulted in more fip distributing to the membrane fraction. such data support the hypothesis that fip is a phosphoprotein. as a control for these experiments, we examined the distribution of another rab effector, rab coupling protein /fip the distribution of rcp/fip between membrane and cytosolic fractions was not altered during the cell cycle. similarly, the levels of phosphorylated rcp/fip did not differ between metaphase and telophase. such data suggest that the cell cycle-induced changes in localization and phosphorylation are specific to fip these data suggest that dephosphorylation of fip accompanies its redistribution from a cytosolic fraction to a membrane fraction. several lines of argument suggest that dephosphorylation of fip alone is unlikely to be sufficient to mediate this. firstly, we and others have shown that fip is recruited to membranes via interaction with rab. indeed, inspection of the data in figure reveals that a fip mutant unable to bind rab is cytosolic. hence, the interaction with rab is likely to be the most important factor controlling fip membrane distribution. secondly, recombinant fip does not bind either to phospholipid vesicles or to intracellular membranes isolated from hela cells. finally, neither s > d nor s > a mutants exhibited altered distribution of gfp-fip throughout the cell cycle. collectively, these data suggest that although fip is a cell cycle-regulated phosphoprotein, the consequences of phosphorylation are not primarily to control the localisation of fip this is also consistent with the location of the phospho-sites within fip3: all four sites studied here are located towards the n-terminal domain of fip and are not within the coiled-coil domain which encompasses the sites of rab arf and cyk- interaction. it should be noted that fip has multiple other potential phospho-acceptor sites, and over such sites have been reported to be phosphorylated in vitro. some of these sites are located within the coiled-coil domain, and may modulate the interaction of fip with other proteins or possibly control fip homo-dimerisation. mutation of the sites reported here did not in our hands modulate the distribution of dynein, nor affect the re-distribution of recycling endosomes characteristic of fip over-expression. similarly, traffic of cargo into and out of the clustered endosomes was not modulated by either s > a or s > d mutation of these sites singly or collectively.  here we have shown that fip is a cell cycle regulated phosphoprotein, and that fip dephosphorylation occurs concomitantly with the shift from largely cytosolic to membrane bound observed as cells pass from metaphase to telophase. the phospho-acceptor sites studied here do not appear to control these changes. there are several possible explanations for this. first, the sites identified are not involved in this interaction, or that multiple sites of de/phosphorylation are involved. alternatively, it is possible that multiple mechanisms of fip targeting exist: this is consistent with studies showing that rab arf and ect are all binding partners for fip and all may contribute to fip localisation. it is unlikely that a single site of phosphorylation may control all of these interactions. we favour a model in which the membrane association of fip is driven mainly by interaction with rab and that dephosphorylation of fip may represent a distinct regulatory mechanism, perhaps involving motor protein interactions resulting in the accumulation of fip in midbodies. further work will be required to resolve these models. 
fip also controls recycling endosome distribution during interphase. finally, we show that de/phosphorylation of the phospho-acceptor sites identified here is not required for the spatial control of recycling endosome distribution or function. in an attempt to identify a functional role for fip phosphorylation, we report that the change in fip distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of fip however, the phospho-acceptor sites identified here did not control this change in distribution. s is dephosphorylated during telophase; mutation of s exerts a modest effect on cytokinesis. we identify four sites of phosphorylation of fip in vivo, s- s- s- and s- and identify s- as a target for cdk1-cyclin b in vitro. rab and its effector molecule, rab11-fip, associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. over-expression of fip3-s102d increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. mutation of s- s- or s- or other previously identified phospho-acceptor sites was without effect on binucleate cell formation and did not modulate the distribution of fip during the cell cycle. our data thus identify fip as a cell cycle regulated phosphoprotein and suggest dephosphorylation of fip accompanies its translocation from the cytosol to membranes during telophase.
 rab and its effector molecule, rab11-fip, associate with recycling endosomes and traffic into the furrow and midbody of cells during cytokinesis. fip also controls recycling endosome distribution during interphase. here, we examine whether phosphorylation of fip is involved in these activities.  we identify four sites of phosphorylation of fip in vivo, s- s- s- and s- and identify s- as a target for cdk1-cyclin b in vitro. of these, we show that s- is phosphorylated in metaphase and is dephosphorylated as cells enter telophase. over-expression of fip3-s102d increased the frequency of binucleate cells consistent with a role for this phospho-acceptor site in cytokinesis. mutation of s- s- or s- or other previously identified phospho-acceptor sites was without effect on binucleate cell formation and did not modulate the distribution of fip during the cell cycle. in an attempt to identify a functional role for fip phosphorylation, we report that the change in fip distribution from cytosolic to membrane-associated observed during progression from anaphase to telophase is accompanied by a concomitant dephosphorylation of fip however, the phospho-acceptor sites identified here did not control this change in distribution.  our data thus identify fip as a cell cycle regulated phosphoprotein and suggest dephosphorylation of fip accompanies its translocation from the cytosol to membranes during telophase. s is dephosphorylated during telophase; mutation of s exerts a modest effect on cytokinesis. finally, we show that de/phosphorylation of the phospho-acceptor sites identified here is not required for the spatial control of recycling endosome distribution or function. cytokinesisrab11-fip3cdk1endosomes 