falciparum clone 3d7 was 
  cultured , and merozoites were purified according to previously described 
  methods ( 17 ) .
invasion 
  inhibition assays were carried out by the addition of k252a ( biomol ) to late 
  stage schizonts at a starting parasitaemia of 0.5% , with 2% hematocrit .
the 
  solvent used was 10% me2so , and inhibitor was added to the 
  appropriate final concentration with a final me2so concentration of 
  0.5% .
invasion rates were measured using a facscalibur machine ( becton 
  dickinson ) 48 h after the addition of inhibitor according to previously 
  described methods ( 18 ) .
these 
  data were confirmed by microscopic examination of giemsa - stained smears of 
  parasite cultures .
total parasite lysates were prepared by the addition of reducing sds - page 
  sample buffer to cell pellets , followed by heating the sample to 95 c for 
  5 min .
carbonate extracts of merozoites were prepared by incubating merozoites 
  in 0.1 m na2co3 , ph 11.0 , on ice for 20 min . 
 
supernatants were collected by ultra - centrifugation at 100,000  
  g in a beckman tl-100 ultracentrifuge .
the carbonate - insoluble pellet 
  was resuspended in reducing sds - page sample buffer . for immunoblots , 
 
carbonate - soluble and insoluble fractions were separated by sds - page on a 12% 
  bistris nupage gel ( invitrogen ) , transferred to protran membrane ( whatman ) , 
  and incubated with the appropriate antibodies .
open reading frames for 
  pfcdpk1 , pfmtip , pfgap50 , and pfgap45 were amplified from late schizont cdna 
  and cloned into the expression vectors pet30xalic , ( pfcdpk1 and pfmtip ; for 
  fig .
3 only ) , pet46eklic 
  ( pfmtip , in all experiments barring those in 
  fig .
pfcdpk1 was expressed in bl21(de3 ) plyss cells , pfmtip , 
  and pfgap50 in bl21(de3 ) cells and pfgap45 in bl21 cells ( all stratagene ) . 
 
after induction with 1 mm isopropyl 
  -d - thiogalactopyranoside , the cells were lysed in bugbuster 
  ( novagen ) and his - tagged proteins purified on nickel - nitrilotriacetic 
  acid - agarose ( qiagen ) in the cases of cdpk1 , gap50 , and mtip and talon resin 
  ( clontech ) in the case of gap45 .
. a kinase - dead mutant of cdpk1 was produced in which asn substituted 
  asp of the catalytic triad .
this was achieved using a quikchange 
  ii mutagenesis kit ( stratagene ) according to the manufacturer 's instructions 
  with the primers : 5-cataaacataatattgtacatcgaaatattaaaccag-3 and 
  5-ctggtttaatatttcgatgtacaatattatgtttatg-3. similarly , 
  recombinant mtip with single amino acid substitutions ( ser , 
  ser , ser , ser , and ser 
  substituted with alanine ) or double s47a / s51a substitutions were generated 
  using a quikchange ii mutagenesis kit ( stratagene ) .
a fragment encoding the calmodulin - like domain of cdpk1 was also amplified 
  using the primers 
  5-gacgacgacaagatggcaacaatattaataaaagtgatcaa-3 and 
  5-gaggagaagcccggtttatgaagatttattatcacaaattttgtg-3 , 
  where the bases in italics represent vector - specific sequence to generate 
  overhangs for ligation - independent cloning into pet46eklic .
the antisera were raised in balb / c 
  mice and new zealand white rabbits for each of the purified recombinant 
  proteins , using standard procedures .
other antibodies used were rabbit 
  anti - ptramp ( 19 ) , mouse 
  monoclonal 89.1 against msp1 
  ( 20 ) , rabbit anti - msp7 
  ( 21 ) , and rabbit anti - pfcrt 
  ( from the malaria reagent and research resource centre ( mr4 ) ) . immunofluorescence 
thin smears of parasites were air - dried 
  and then fixed using 3% paraformaldehyde in phosphate - buffered saline ( pbs ) 
  for 30 min at room temperature .
triton 
  x-100 in pbs for 10 min followed by blocking in 3% bovine serum albumin in pbs 
  overnight at 4 c .
antibodies were diluted in blocking buffer and 
  incubated on the slide for 1 h followed by washing in pbs .
antigen 
  visualization was achieved using alexa - fluor-488- or 594-conjugated 
  anti - species antibodies ( molecular probes ) .
the images were processed using a 
  deltavision cooled charge - coupled device imaging system ( applied precision 
  inc . ) .
images from the fluorescence microscope were collected and analyzed 
  with softworx and prepared for publication with adobe photoshop .
cell lysis and shrimp alkaline phosphatase 
  treatment  synchronized schizonts ( 40 - 45 h ) were lysed in 0.15% 
  saponin in tris - buffered saline .
parasites were lysed in a buffer containing 7 
  m urea , 2 m thiourea , 4% chaps , 25 mm tris 
  ( all plusone reagents ; ge healthcare ) , using 250 l/1  
  10 cells . to ensure complete lysis ,
the cells were frozen in a dry 
  ice / ethanol bath and thawed at 29 c five times .
an alkaline phosphatase reaction was set up using 
  300 g of cell lysate , 3 l of protease inhibitor mix ( ge healthcare ) , 
  20 units of shrimp alkaline phosphatase ( sap ; promega ) , in a total of 300 
  l .
a control reaction was set up in parallel as above but with the 
  addition of 6 l of phosphatase inhibitor mixture i ( sigma ) and 20 
  mm imidazole .
the lysates were incubated at 37 c for 30 min 
  and then heated to 65 c for 15 min to inactivate the sap .
the proteins 
  were precipitated using methanol / chloroform , followed by an acetone 
  precipitation , and resuspended in rehydration buffer containing 6 m 
  urea , 2 m thiourea , and 4% chaps .
two - dimensional electrophoresis and western 
  blotting  rehydration buffer containing 0.01% dithiothreitol , 0.002% 
  bromphenol blue and 0.02% immobilized ph gradient buffer was added to 50 g 
  of control treated or sap treated schizont lysate to give a total volume of 
  125 l / sample .
isoelectric focusing was carried out on ipgphor ( ge 
  healthcare ) using 7-cm immobilized ph gradient strips ( ph 3 - 10nl ; ge 
  healthcare ) to a maximum voltage of 5000 v , collecting a total of 22000 vh . 
 
after isoelectric focusing , immobilized ph gradient strips were incubated for 
  15 min at room temperature in equilibration buffer ( 2% sds , 50 mm 
  tris - hcl , ph 8.8 , 6 m urea , 30% ( v / v ) glycerol , 0.002% bromphenol 
  blue ) with 10 mg / ml dithiothreitol , followed by a second incubation in 
  equilibration buffer containing 25 mg / ml iodoacetamide .
the strips were run in 
  parallel in the second dimension using nupage 4 - 12% bistris zoom gels 
  ( invitrogen ) .
the gels were blotted onto polyvinylidene difluoride membrane 
  and probed with affinity - purified rabbit anti - mtip ( 4 g / ml ) or polyclonal 
  rabbit anti - gap45 ( 1/500 ) , using an horseradish peroxidase - conjugated 
  anti - rabbit igg ( 1/15,000 ; sigma ) as a secondary antibody .
the signal was 
  detected using supersignal west pico chemiluminescent substrate ( pierce ) . 
 
samples from a parasite time course were lysed in the same buffer but 
  separated by one - dimensional sds - page .
scanning densitometry of 
  autoradiographs was performed using image j software 
  ( 22 ) .
kinase assays and kinetics  radiolabeled phosphate 
  incorporation kinase assays were carried out with 15 pmol of cdpk1 and 200 
  pmol of substrate protein in 20 mm tris - hcl , ph 8.0 , 20 
  mm mgcl2 , 100 m atp , 0.1 mbq of 
  [ -p]atp , 1 mm cacl2 .
the reactions 
  were incubated for 15 min at 30 c , stopped by the addition of reducing 
  sample buffer , and analyzed by sds - page .
after fixation in a solution of 30% 
  methanol plus 5% glycerol , the gels were dried and exposed to biomaxmr film 
  ( kodak ) . to prepare samples for mass spectrometry and fluorimetric analysis , 
  3.5 nmol of substrate proteins were incubated with 150 pmol of cdpk1 under the 
  same conditions ( without radiolabeled atp ) for 16 h at room temperature .
kinase activity measurements were made using a continuous 
  spectrophotometric assay based on one described by cook et al . 
 
a typical assay 
  contained the following components in a total volume of 0.5 ml in a 
  10-mm - pathlength cuvette : 50 mm hepes ( ph 7.5 ) , 1 mm 
  cacl2 , 10 mm mgcl2 , 6 units of pig heart 
  l - lactic dehydrogenase , 4 units of rabbit muscle pyruvate kinase , 1 
  mm phosphoenolpyruvate , 250 mm nadh .
concentrations of 
  atp and mtip ( or gap45 ) were varied as appropriate . in experiments for 
  determining the km for mtip and gap45 ,
the reactions were initiated by the 
  addition of cdpk1 ( at 10 - 15 g / ml ) , and the rates ( at 30 c ) were 
  obtained by recording absorbance changes at 340 nm .
kinetic 
  parameters were determined by direct nonlinear least squares fits to the 
  michaelis - menten equation using curfit software ( dr .
this 
  coupled assay was validated by comparison with a radio - metric assay using 
  [ -p]atp 
  ( 24 ) .
n - octyl--d - glucopyranoside , 50 mm 
  tris - hcl , 5 mm edta , 150 mm nacl , ph 8.0 , on ice for 20 
  min .
the supernatants were collected by ultracentrifugation at 100,000  
  g in a beckman tl-100 ultracentrifuge .
gap45 was purified from these 
  supernatants using an immunoaffinity column of rabbit anti - gap45 antibodies 
  cross - linked to cyanogen bromide - activated sepharose ( ge healthcare ) .
after 
  extensive washing with lysis buffer and pbs , gap45 was eluted using 0.1 
  m glycine , ph 2.7 , and separated by sds - page , and the relevant band 
  was excised from the gel .
the gel was dried and reswollen in a 
  sufficient volume to cover of 2 ng/l modified sequencing grade trypsin or 
  1 ng/l aspn ( both promega ) in 5 mm ammonium bicarbonate .
after 
  overnight digestion at 32 c , the supernatant was acidified by the 
  addition of a 1/10th volume of 4% trifluoroacetic acid .
purification of phosphopeptides  recombinant proteins were 
  phosphorylated by cdpk1 , subjected to digestion with trypsin ( mtip ) or aspn 
  ( gap45 ) and phosphopeptides enriched by purification on either gallium spin 
  columns ( pierce ) for gap45 or talon magnetic beads ( clontech ) for mtip 
  according to the manufacturer 's instructions .
peptide mass fingerprinting by maldi - tof mass 
  spectrometry  peptides from protease digestion were analyzed by 
  maldi - tof , and the spectra were examined to identify phosphorylated peptides . 
 
peptide mass fingerprinting was performed using a reflex iii maldi 
  time - of - flight mass spectrometer ( bruker daltonik gmbh , bremen , germany ) , 
  equipped with a nitrogen laser and a scout-384 probe , to obtain positive ion 
  mass spectra of digested protein with pulsed ion extraction in reflectron 
  mode . an accelerating voltage of 26 kv was used with detector bias gating set 
  to 2 kv and a mass cut - off of m / z 650 .
the matrix surfaces 
  were prepared using recrystallized -cyano-4-hydroxycinammic acid and 
  nitrocellulose using the fast evaporation method 
  ( 25 ) .
0.4 l of acidified 
  digestion supernatant was deposited on the matrix surface and allowed to dry 
  prior to desalting with water .
expression of pfcdpk1 in merozoites and localization to the parasite 
  plasma membrane  we used anti - cdpk1 antibodies in a western blot 
  with schizont and merozoite cell lysates .
it is evident that pfcdpk1 is 
  present in free merozoites , as well as schizonts 
  ( fig .
it was suggested that pfcdpk1 is located in the pvm 
  of developing parasites ( 8) , 
  but because free merozoites do not contain a pvm , it is clear that cdpk1 must 
  be present in another location in the parasite .
the protein from merozoites is 
  still associated with membranes , as demonstrated by partitioning of cdpk1 in 
  the insoluble fraction of merozoites extracted with a high ph carbonate buffer 
  ( fig .
the same 
  fraction contains membrane - associated proteins such as ptramp 
  ( 19 ) .
by contrast , peripheral 
  membrane proteins such as msp7 are solubilized by this buffer 
  ( fig .
b , western blots of merozoites 
  extracts using anti - cdpk1 , anti - ptramp , and anti - msp7 antibodies .
lanes 
  1 , total merozoite lysate ; lanes 2 , carbonate buffer soluble 
  fraction ; lanes 3 , carbonate buffer insoluble fraction .
the 
  numbers on the left of each panel indicate the positions of 
  molecular mass markers in kilodaltons .
b , western blots of merozoites 
  extracts using anti - cdpk1 , anti - ptramp , and anti - msp7 antibodies .
lanes 
  1 , total merozoite lysate ; lanes 2 , carbonate buffer soluble 
  fraction ; lanes 3 , carbonate buffer insoluble fraction .
the 
  numbers on the left of each panel indicate the positions of 
  molecular mass markers in kilodaltons .
a , an 
  anti - cdpk1 antibody detects cdpk1 at the parasite periphery in schizonts 
  ( panel i ) and free merozoites ( panel ii ) .
the second 
  panel shows a merged image of nuclear staining with 
  4,6-diamino-2-phenylindole ( blue ) and the bright field 
  image , and the third panel shows the bright field image .
b , panel 
  i , cdpk1 colocalizes with mtip around the periphery of merozoites but is 
  also found around the residual body , marked with an arrowhead , from 
  which mtip is absent . in young ( panel ii ) and segmented ( panel 
  iii ) schizonts , cdpk1 colocalizes with the plasma membrane marker msp1 . 
 
both proteins are also detected around the residual body , marked with an 
  arrowhead .
cdpk1 does not colocalize with the food vacuole marker 
  pfcrt in either immature ( panel iv ) or mature ( panel v ) 
  schizonts .
the third panels in all of the images show the merged 
  images of the antibody staining , beside which is a merged image of the bright 
  field image and the nuclei of the parasites stained with 
  4,6-diamino-2-phenylindole ( blue ) , followed by the 
  bright field image alone .
c , both cdpk1 ( red ) and msp1 
  ( green ) can be detected on residual bodies that are released upon 
  schizont rupture .
the merged images of the antibody staining are shown in the 
  third panels , followed by the bright field image . in all cases
the 
  white scale bar in the first panel of each set of images represents 1 
  m .
a , an 
  anti - cdpk1 antibody detects cdpk1 at the parasite periphery in schizonts 
  ( panel i ) and free merozoites ( panel ii ) .
the second 
  panel shows a merged image of nuclear staining with 
  4,6-diamino-2-phenylindole ( blue ) and the bright field 
  image , and the third panel shows the bright field image .
b , panel 
  i , cdpk1 colocalizes with mtip around the periphery of merozoites but is 
  also found around the residual body , marked with an arrowhead , from 
  which mtip is absent . in young ( panel ii ) and segmented ( panel 
  iii ) schizonts , cdpk1 colocalizes with the plasma membrane marker msp1 . 
 
both proteins are also detected around the residual body , marked with an 
  arrowhead .
cdpk1 does not colocalize with the food vacuole marker 
  pfcrt in either immature ( panel iv ) or mature ( panel v ) 
  schizonts .
the third panels in all of the images show the merged 
  images of the antibody staining , beside which is a merged image of the bright 
  field image and the nuclei of the parasites stained with 
  4,6-diamino-2-phenylindole ( blue ) , followed by the 
  bright field image alone .
c , both cdpk1 ( red ) and msp1 
  ( green ) can be detected on residual bodies that are released upon 
  schizont rupture .
the merged images of the antibody staining are shown in the 
  third panels , followed by the bright field image . in all cases
the 
  white scale bar in the first panel of each set of images represents 1 
  m .
we examined the subcellular location of cdpk1 in free merozoites and 
  schizonts by immunofluorescence .
cdpk1 is present at the periphery of 
  merozoites , both within schizonts , and released 
  ( fig .
however , dual staining 
  of schizonts with anti - cdpk1 antibodies and antibodies raised against mtip ( a 
  marker for the imc ) and msp1 ( a marker for the parasite plasma membrane ) 
  revealed interesting differences .
cdpk1 colocalizes with mtip around 
  developing merozoites in segmented schizonts but is also present in membranes 
  around the mature food vacuole / residual body of the schizonts , whereas mtip is 
  absent ( fig .
in young schizonts 
  ( 39 h post - invasion ) where segmentation has not begun , both cdpk1 and 
  msp1 are present in the parasite plasma membrane 
  ( fig .
both cdpk1 
  and msp1 are seen in the plasma membrane around the food vacuole 
  ( fig .
2b , panel 
  iii ) . to exclude the possibility that cdpk1 is present within the food 
  vacuole membrane , we stained parasites with antibodies against pfcrt , a known 
  marker of the food vacuole membrane .
there was no coincidence of staining in 
  early schizonts and a small amount of colocalization in segmented schizonts 
  ( fig .
we also see cdpk1and msp1 in the membranes 
  surrounding residual bodies , structures that consist of food vacuoles 
  containing hemozoin that are discarded when a schizonts ruptures .
residual 
  bodies are surrounded by parasite plasma membrane , a consequence of the 
  mechanism by which merozoites are pinched off from the syncytium within the 
  erythrocyte ( 26 , 
  27 ) and therefore stain with 
  known markers of the plasma membrane such as msp1 
  ( fig .
gap45 and mtip are substrates for pfcdpk1 in vitro  because 
  of its close proximity to the imc and its similar membrane anchorage to gap45 , 
  a known component of the motor complex in the imc
, we tested the ability of 
  recombinant cdpk1 to phosphorylate gap45 , gap50 , and mtip in vitro . 
 
initially , kinase assays were performed in which the incorporation of 
  p from radiolabeled atp into substrate proteins was measured .
the 
  proteins were separated by sds - page , and phosphorylation was detected by 
  autoradiography .
both mtip and gap45 were efficiently phosphorylated by cdpk1 , 
  whereas another component of the motor complex , gap50 , was not .
further 
  control proteins , msp722 , and bovine serum albumin were also not 
  phosphorylated by cdpk1 ( fig
the absolute calcium dependence of this 
  phosphorylation and the inability of a  kinase - dead  mutant of 
  cdpk1 to autophosphorylate or modify mtip is shown in 
  fig .
the 
  kinase - dead mutant was made by modifying asp in the hrd 
  catalytic triad of cdpk1 to asn .
we saw no effect of mtip or gap45 on 
  autophosphorylation of cdpk1 , in contrast to the increased autophosphorylation 
  of cdpk1 in the presence of rkip 
  ( 10 ) .
further coupled - enzyme 
  kinase assays were used to determine the kinetic parameters for the enzyme 
  substrate pairs .
we determined the km for mtip to be 9 
   0.9 m ( fig . 
 
the km calculation for gap45 was 
  carried out with a somewhat restricted data set because of limiting amounts of 
  gap45 ; however , a km of 18  5 m was 
  calculated ( fig .
the km of cdpk1 for atp was calculated to be 125  
  13 m for mtip and 96  28 m for gap45 
  ( fig .
the activity of cdpk1 was calculated to be 0.2 
  mol min mg enzyme with mtip as a substrate and 
  0.5 mol min mg enzyme with gap45 as a substrate . 
 
previous calculations of cdpk1 activity toward different substrates have been 
  made . when phosphorylating the exogenous substrate casein , cdpk1 had an 
  activity of 0.28 mol min mg enzyme , whereas with 
  the parasite protein rkip as a substrate , the activity was 0.04 mol 
  min mg enzyme 
  ( 10 ) .
a , the upper 
  panel shows a coomassie blue - stained gel of an in vitro kinase 
  assay performed with recombinant cdpk1 and mtip , gap45 , bovine serum albumin , 
  msp722 , or gap50 substrates .
the lower panel is an 
  autoradiograph of duplicate reactions incubated in the presence of 
  [ -p]atp . only mtip and gap45 are phosphorylated by cdpk1 . 
  in all cases
we see autophosphorylation of cdpk1 , except when egta is included 
  in the reaction mixture .
recombinant gap50 was treated with factor xa to 
  remove the 12-kda thioredoxin tag encoded by the pet32 vector .
residual factor 
  xa has cleaved a proportion of the tag present on cdpk1 ( 5 kda , encoded 
  in the pet30 vector ) , resulting in cdpk1 running as a doublet in this lane . 
 
b , phosphorylation of mtip is absolutely dependent on the presence of 
  calcium , and a substitution of asn for asp in the catalytic 
  triad of cdpk1 results in an inactive protein .
the two upper panels 
  show coomassie - stained gels of kinase reactions ; the left panel shows 
  reactions with wild - type cdpk1 , and the right panel shows reactions 
  with the asp to asn substitution .
the lower panels show 
  autoradiographs of the same reactions incubated in the presence of 
  [ -p]atp .
a , the upper 
  panel shows a coomassie blue - stained gel of an in vitro kinase 
  assay performed with recombinant cdpk1 and mtip , gap45 , bovine serum albumin , 
  msp722 , or gap50 substrates .
the lower panel is an 
  autoradiograph of duplicate reactions incubated in the presence of 
  [ -p]atp .
we see autophosphorylation of cdpk1 , except when egta is included 
  in the reaction mixture .
recombinant gap50 was treated with factor xa to 
  remove the 12-kda thioredoxin tag encoded by the pet32 vector .
residual factor 
  xa has cleaved a proportion of the tag present on cdpk1 ( 5 kda , encoded 
  in the pet30 vector ) , resulting in cdpk1 running as a doublet in this lane . 
 
b , phosphorylation of mtip is absolutely dependent on the presence of 
  calcium , and a substitution of asn for asp in the catalytic 
  triad of cdpk1 results in an inactive protein .
the two upper panels 
  show coomassie - stained gels of kinase reactions ; the left panel shows 
  reactions with wild - type cdpk1 , and the right panel shows reactions 
  with the asp to asn substitution .
the lower panels show 
  autoradiographs of the same reactions incubated in the presence of 
  [ -p]atp .
enzyme - coupled kinase 
  assays measuring the reduction in nadh ( a340/min ) 
  are shown for cdpk1 while varying the substrate concentration . a , 
  mtip ; b , gap45 .
the atp concentration in both cases was 700 
  m . from these the km for mtip is calculated 
  to be 9  0.9 m , and that for gap45 is 18  5 
  m .
a340/min plotted against atp 
  concentration , to give the km for atp of cdpk1 with mtip 
  ( c ) or gap45 ( d ) as 125  13 and 96  28 
  m , respectively .
these experiments were carried out at constant 
  substrate concentration ( 8.2 m mtip or 3.5 m 
  gap45 ) . in all cases data points (  )
are plotted , with the solid 
  line representing the best fit curve calculated by direct nonlinear least 
  squares fit to the michaelis - menten equation using in - house software .
enzyme - coupled kinase 
  assays measuring the reduction in nadh ( a340/min ) 
  are shown for cdpk1 while varying the substrate concentration . a , 
  mtip ; b , gap45 .
from these the km for mtip is calculated 
  to be 9  0.9 m , and that for gap45 is 18  5 
  m .
a340/min plotted against atp 
  concentration , to give the km for atp of cdpk1 with mtip 
  ( c ) or gap45 ( d ) as 125  13 and 96  28 
  m , respectively .
these experiments were carried out at constant 
  substrate concentration ( 8.2 m mtip or 3.5 m 
  gap45 ) . in all cases data points (  )
are plotted , with the solid 
  line representing the best fit curve calculated by direct nonlinear least 
  squares fit to the michaelis - menten equation using in - house software .
metastable assignments were confirmed by post - source decay 
  analysis ( data not shown ) .
recombinant proteins were phosphorylated by cdpk1 
  and subjected to digestion with trypsin ( mtip ) or aspn ( gap45 ) , and 
  phosphopeptides were enriched by purification on either gallium spin columns 
  or talon magnetic beads .
a single peptide from mtip was detected , 
  kplsieesfenseesees - vadiqqleek , which spans amino acids 44 - 71 in the mtip 
  sequence .
the peptides were isolated in which either one or two of the five 
  serines ( ser , ser , ser , 
  ser , and ser ) were phosphorylated , along with their 
  metastable products , because of phosphoric acid loss 
  ( fig .
two phosphopeptides and their metastable products were identified , each 
  containing a single phosphorylated residue .
the gap45 phosphopeptides are 
  dyatqenksfeekhle and dlersnsdiysesqkf , spanning amino acids 81 - 96 and 97 - 112 , 
  respectively ( fig . 
 
the gap45 spectrum also shows a phosphorylated 
  peptide ( m / z 1934.829 ) that is derived from the vector tag 
  of pqe60 . to identify the specific serine residues in mtip that are phosphorylated by 
  cdpk1
, recombinant mtip proteins were made where serine residues 47 , 51 , 55 , 
  58 , and 61 were individually mutated to alanine by site - directed mutagenesis . 
 
radio - metric kinase assays were performed , and the incorporation of 
  p into the proteins was compared with that of the wild - type mtip 
  protein ( fig .
5c ) . 
  substitution of serines 55 , 58 , and 61 did not result in a significant 
  reduction in phosphorylation of mtip by cdpk1 .
in contrast , mutation of 
  ser or ser resulted in a 73 and 34% reduction in the 
  incorporation of p , respectively . when a double s47a / s51a mutant 
  of mtip was used , a reduction of p incorporation of 90% was 
  observed .
these data suggest that the primary phosphorylation of mtip by cdpk1 
  occurs on ser , with a secondary phosphorylation occurring 
  nonstoichiometrically on ser .
because the signal in the assay did 
  not drop to zero when the s47a / s51a mtip mutant was used , it would seem that 
  there is a third site in mtip that is phosphorylated at a much lower 
  efficiency that remains unidentified .
mtip and gap45 are phosphoproteins in the parasite  it is 
  crucial to establish that both gap45 and mtip are phosphorylated in parasites . 
  to this end
, schizont extracts were separated by two - dimensional sds - page and 
  then immunoblotted with antibodies to either mtip or gap45 .
when phosphate groups were removed 
  by treating the lysate with sap , mtip migrates as a single spot 
  ( fig .
when the same extracts were probed using an anti - gap45 antibody , 
  gap45 was identified as three spots that differ in both mass and charge in the 
  mock treated lysate ( fig . 
 
cell lysates from 
  parasites ranging from late trophozoite ( 33 h post - invasion ) to late schizont 
  stages ( 45 h post - invasion ) were separated by one - dimensional sds - page and 
  blotted with an anti - gap45 antibody .
expression of gap45 is just detectable at 
  early time points , and its expression peaks at 45 h post - invasion , where it 
  migrates as a doublet . the upper band ( containing phosphorylated gap45 ) 
 
increases in intensity in relation to the lower band ( nonphosphorylated gap45 ) 
  as development of the parasite progresses 
  ( fig .
if the amount of protein loaded is adjusted to normalize gap45 
  levels , the relative proportion of phosphorylated versus 
  nonphosphorylated gap45 at a given time point can be calculated 
  ( fig .
the proportion of phosphorylated gap45 increases 
  from 12% at 33 h to 62% at 45 h. this increase mirrors the expression profile 
  of cdpk1 ( fig .
figure 5.analysis of phosphopeptides from recombinant mtip and gap45 . 
 a , mtip tryptic peptides eluted from talon magnetic beads .
a single 
  peptide is seen , with either one or two phosphates , along with metastable 
  post - source decay products .
peptide 1 is derived 
  from the vector - encoded protein tag , whereas peptides 2 and 3 are from gap45 . 
 
c , incorporation of p into mtip mutants was measured by 
  scanning densitometry of autoradiographs of in vitro kinase assays 
  and compared with wild - type protein ( wt ) .
the s47a mutant showed 
  p incorporation of 27% , the and s51a mutant showed 66% compared 
  with wild - type mtip .
mutation of ser , ser , and 
  ser did not significantly affect incorporation of p. 
  a double mutant ( s47a / s51a ) gave a signal of 10% compared with wt mtip .
a single 
  peptide is seen , with either one or two phosphates , along with metastable 
  post - source decay products .
peptide 1 is derived 
  from the vector - encoded protein tag , whereas peptides 2 and 3 are from gap45 . 
 
c , incorporation of p into mtip mutants was measured by 
  scanning densitometry of autoradiographs of in vitro kinase assays 
  and compared with wild - type protein ( wt ) .
the s47a mutant showed 
  p incorporation of 27% , the and s51a mutant showed 66% compared 
  with wild - type mtip .
mutation of ser , ser , and 
  ser did not significantly affect incorporation of p. 
  a double mutant ( s47a / s51a ) gave a signal of 10% compared with wt mtip .
purified protein was excised from an 
  sds - page gel and digested with either aspn or trypsin , and the resultant 
  peptides were analyzed by maldi - tof to identify phosphorylated peptides .
two 
  phosphopeptides were identified : dyatqenksfeekhle in the aspn digested sample 
  ( fig . 7a ) and an 
  additional tryptic peptide lsepaheesiyftyr corresponding to residues 141 - 155 
  ( fig .
the aspn 
  peptide is identical to one of the peptides identified in in vitro 
  phosphorylation of gap45 by cdpk1 .
the detection of a phosphopeptide in 
  parasite - derived gap45 that was absent from the tryptic digest of recombinant 
  gap45 phosphorylated by cdpk1 ( data not shown ) is strong evidence that an as 
  yet unidentified parasite protein kinase phosphorylates this site .
inhibition of cdpk1 by the bisindolocarbazole k252a  the 
  effect of the kinase inhibitor kt5926 has been tested in t. gondii . 
 
the inhibitor blocked attachment of parasites to host cells and reduced the 
  activity of tgcdpk1 with similar ic50 values of 100 
  nm ( 11 ) .
we tested 
  the effect of a related inhibitor , k252a , on p. falciparum cultures 
  and on the ability of cdpk1 to phosphorylate recombinant mtip in 
  vitro .
k252a potently inhibited cdpk1 activity in vitro , with an 
  ic50 of 45 nm .
falciparum invasion of 
  erythrocytes was inhibited in the presence of k252a . at very high 
  concentrations (
0.8 - 5 m ) invasion of erythrocytes was 
  completely blocked . on examination of giemsa - stained smears of the culture , it 
  was clear that the egress of merozoites from schizonts was blocked rather than 
  invasion of erythrocytes by merozoites . at intermediate concentrations of 
  k252a ( 0.4 m ) ,
invasion was inhibited by 65% . at these 
  intermediate concentrations of k252a , egress of merozoites from schizonts
was 
  not prevented ; however , invasion of erythrocytes by merozoites was 
  significantly inhibited , with half - maximal inhibition occurring at 348 
  nm ( fig . 8) .
we have shown that cdpk1 is present in both schizonts and free merozoites 
  in a membrane - bound fraction , which is consistent with the dual acylation of 
  the protein .
the protein is located at the plasma membrane of the developing 
  intracellular schizont both before and after segmentation as indicated by 
  colocalization with msp1 . in segmenters
it can be detected on the plasma 
  membrane that surrounds the food vacuole , which is identified by the presence 
  of the hemozoin pigment and reactivity with antibodies to crt 
  ( 28 , 
  29 ) .
notably , mtip , a marker 
  for the imc , is absent from this membrane .
following schizont rupture and 
  release of merozoites , the protein is still detected at the periphery of the 
  discarded residual body derived from the food vacuole but most importantly is 
  located at the periphery of the free merozoite
. in free merozoites we can not 
  exclude that it is also present on other peripheral membranes , particularly 
  the imc .
it has previously been proposed that cdpk1 is exported from the 
  schizont to the pvm of the intracellular parasite , which would require 
  transfer of this protein across the parasite plasma membrane followed by 
  insertion into the pvm ( 8) . 
 
although we can not exclude that this transfer occurs , our results indicate 
  that cdpk1 is largely present in a membrane bound form at the periphery of 
  both schizonts and free merozoites .
we suggest therefore that in free 
  merozoites the protein is located in the space between the plasma membrane 
  ( pm ) and the inner membrane complex ( imc ) , which is the location of the 
  actomyosin motor that drives merozoite invasion of red blood cells 
  ( 30 ) .
the dual acylation of 
  cdpk1 is sufficient to retain it at the membrane , and this post - translational 
  modification is identical to that of gap45 , which is also myristoylated and 
  palmitoylated at the n terminus 
  ( 13 ) . it is noteworthy that 
  gap45 , which is a component of the motor complex , is thought to be located in 
  the space between the imc and the pm but attached to the imc membrane 
  ( 12 , 
  31 ) ; it may be that cdpk1 can 
  be associated with both the inner face of the pm and the outer face of the 
  imc .
a , 
  protein extracted from late - stage schizonts was subjected to treatment with 
  sap , separated by two - dimensional sds - page and western blotted with anti - mtip 
  or anti - gap45 antibodies .
both mtip ( panel ii ) and gap45 ( panel 
  iv ) antibodies detect only a single spot . in mock - treated samples ,
two 
  spots are visible in the anti - mtip blot ( panel i ) and three spots in 
  the anti - gap45 blot ( panel iii ) .
the phosphorylated proteins that 
  disappear upon sap treatment are circled in panels i and 
  iii .
b , lysates from a developmental time - course of 
  parasites were probed with anti - gap45 ( panel i ) and anti - cdpk1 
  antibodies ( panel ii ) .
panel iii , loading of lysates 
  normalized according to gap45 levels shows that the relative proportion of 
  phosphorylated gap45 ( upper band ) increases compared with 
  unphosphorylated gap45 as parasite development progresses .
panel iv , 
  scanning densitometry of anti - gap45 immunoblots showing the proportion of 
  phosphorylated and unphosphorylated gap45 at each developmental time point . 
 
each value represents the average of two experiments , with error bars 
  showing one standard deviation from the mean .
a , 
  protein extracted from late - stage schizonts was subjected to treatment with 
  sap , separated by two - dimensional sds - page and western blotted with anti - mtip 
  or anti - gap45 antibodies . both mtip ( panel ii ) and gap45 ( panel 
  iv )
two 
  spots are visible in the anti - mtip blot ( panel i ) and three spots in 
  the anti - gap45 blot ( panel iii ) .
the phosphorylated proteins that 
  disappear upon sap treatment are circled in panels i and 
  iii .
b , lysates from a developmental time - course of 
  parasites were probed with anti - gap45 ( panel i ) and anti - cdpk1 
  antibodies ( panel ii ) .
panel iii , loading of lysates 
  normalized according to gap45 levels shows that the relative proportion of 
  phosphorylated gap45 ( upper band ) increases compared with 
  unphosphorylated gap45 as parasite development progresses .
panel iv , 
  scanning densitometry of anti - gap45 immunoblots showing the proportion of 
  phosphorylated and unphosphorylated gap45 at each developmental time point . 
 
each value represents the average of two experiments , with error bars 
  showing one standard deviation from the mean .
figure 7.maldi-tof analysis of phosphopeptides from affinity - purified 
  parasite - derived gap45 . phosphopeptides ( p ) and their post - source 
  decay products ( [ m - h3po4 ] ) from aspn 
  digestion ( a ) and trypsin digestion of merozoite - derived gap45 
  ( b ) .
maldi - tof analysis of phosphopeptides from affinity - purified 
  parasite - derived gap45 .
phosphopeptides ( p ) and their post - source 
  decay products ( [ m - h3po4 ] ) from aspn 
  digestion ( a ) and trypsin digestion of merozoite - derived gap45 
  ( b ) .
the 
  activity has an absolute requirement for calcium , and the inactivity of the 
  kinase - dead form of the protein resulting from replacement of asp by asn in 
  the active site is consistent with the detected kinase activity being 
  attributable to cdpk1 . of particular interest
is that the enzyme can 
  phosphorylate two proteins of the motor complex , mtip and gap45 , whereas a 
  third component of the complex , gap50 and other control proteins , are not 
  modified by cdpk1 .
both mtip and gap45 are good substrates for cdpk1 with 
  km values in the low micromolar range that is well within 
  the suggested threshold of a km  50 m 
  for potential substrates ( 1 ) . 
 
similarly the activity of cdpk1 with both mtip and gap45 is in the same range 
  as its activity with the exogenous substrate casein ( 0.2 - 0.5 mol 
  min mg enzyme ) and well above the reported activity 
  with the parasite protein rkip as a substrate ( 0.04 mol min 
  mg enzyme ) ( 10 ) . 
 
these data confirm that p. falciparum cdpk1 is a calcium - dependent 
  protein kinase and show that two components of the merozoite motor complex , 
  mtip and gap45 , are good substrates for phosphorylation . both mtip and gap45 
 
are located in the same cytoplasmic location as cdpk1 , between the pm and the 
  imc .
a , 
  cdpk1 activity on recombinant mtip in the presence of k252a was assayed using 
  radiolabeled atp .
half - maximal inhibition of invasion occurred at 348 nm 
  k252a . in both graphs ,
the values plotted are the averages of triplicate 
  experiments , with the error bars showing one standard deviation from 
  the mean .
. a , 
  cdpk1 activity on recombinant mtip in the presence of k252a was assayed using 
  radiolabeled atp .
in both graphs , the values plotted are the averages of triplicate 
  experiments , with the error bars showing one standard deviation from 
  the mean .
gap45 is directly 
  associated with the outer membrane of the imc , in a complex with mtip , gap50 , 
  and myosin a. gap45 is further phosphorylated by an unidentified kinase in 
  merozoites .
gap45 is directly 
  associated with the outer membrane of the imc , in a complex with mtip , gap50 , 
  and myosin a. gap45 is further phosphorylated by an unidentified kinase in 
  merozoites . to examine the phosphorylation of mtip and gap45 further , we studied the 
  sites of modification by cdpk1 of the recombinant expressed proteins in 
  vitro by peptide digestion and analysis by mass spectrometry . 
 
bioinformatic predictions suggest that many residues in both mtip and gap45 
  are capable of being phosphorylated by serine - threonine protein kinases .
we 
  identified experimentally one peptide from mtip containing five serines that 
  was phosphorylated once or twice and by mutagenesis have shown that the 
  primary phosphorylation occurs at ser , with a minor 
  phosphorylation at ser .
the two phosphopeptides from gap45 
  contain either one threonine and one serine or four serines , respectively , 
  which were both phosphorylated only in one position .
it is unlikely that the 
  threonine residue in gap45 peptide 81 - 96 is phosphorylated because the 
  phosphorylated protein did not react with phospho - threonine specific 
  antibodies ( data not shown ) , and therefore the phosphorylated residue is 
  ser .
the presence of multiple serines in peptides 97 - 112 and 
  141 - 155 of gap45 and poor fragmentation of the peptides in the mass 
  spectrometer have not allowed a definitive identification of the specific 
  residues that are modified .
although we showed that both mtip and gap45 could be phosphorylated in 
  vitro , it was important to show that this also occurred in vivo . 
 
the phosphorylation of both proteins in the parasite was confirmed by 
  two - dimensional gel analysis of the proteins with or without phosphatase 
  treatment . in both cases
multiple forms of the protein in the absence of 
  phosphatase treatment were replaced by a single spot , consistent with the 
  removal of phosphate residues by the enzyme .
at least one of the forms of each 
  of the proteins in the parasite prior to phosphatase treatment migrated in the 
  same location as the enzyme - treated proteins , suggesting that the 
  phosphorylation is only partial and substoichiometric in schizonts .
we have 
  also demonstrated that phosphorylation of gap45 increases as parasite 
  development progresses and that this mirrors cdpk1 expression . to confirm the 
  phosphorylation of the proteins in vivo
, we wished to purify them 
  from parasite extracts and subject them to protease digestion and analysis by 
  mass spectrometry of phosphorylated peptides .
we were unable to purify 
  sufficient mtip for analysis , but sufficient gap45 was obtained to carry out 
  some analyses .
one of the two phosphopeptides observed in recombinant gap45 
  was identified , confirming that this sequence is phosphorylated both in 
  vitro and in vivo and implicating cdpk1 as the kinase 
  responsible .
in addition , a second novel phosphorylated peptide was 
  identified , suggesting that gap45 is also the substrate for a second kinase .
a 
  recent study has shown that gap45 is a substrate of pfpkb in vitro 
  ( 41 ) ; it is possible that 
  pfpkb is responsible for the phosphorylation of gap45 at the 
   non - cdpk1  site identified here .
9 . activation of plasma 
  membrane - situated cdpk1 by a calcium signal results in phosphorylation of 
  gap45 and mtip associated with the imc .
. our data also suggest 
  that a calcium signal of sufficient magnitude to activate cdpk1 exists in 
  merozoites prior to erythrocyte attachment and invasion .
it is of interest to note that the phosphorylated residues in mtip are in 
  the n - terminal region that was not present in the recently described structure 
  ( 32 ) .
the n - terminal 70 
  residues extend beyond the region of mtip that is similar to calmodulin and 
  other myosin light chains and binds the c - terminal tail of myosin a. 
  furthermore , phosphorylation of mtip does not affect its binding to a myoa 
  tail peptide.5 the 
  role of this n - terminal region of mtip is not known , but it is possible that 
  it may be important for binding to gap45 because gap45 is in the motor complex 
  but does not bind directly to the myosin 
  tail.5 thus it is 
  possible that phosphorylation of either or both mtip and gap45 may modulate 
  the interaction between them .
another possibility is that phosphorylation of mtip , which is a myosin 
  light chain , is important in control of the motor .
phosphorylation of the 
  regulatory light chains of myosin ( rlcs ) has been shown to be important in 
  modulating the myosin motor in several systems . in skeletal muscle , 
 
phosphorylation of rlc by ca / calmodulin - dependent skeletal muscle 
  myosin light chain kinase leads to enhanced skeletal muscle contraction 
  ( 33 ) . in cardiac muscle , 
 
phosphorylation of rlc results in increased cross - bridge formation and 
  enhanced force from contraction 
  ( 34 ) . in smooth muscle , 
 
phosphorylation of the rlc of myosin ii by the 
  ca / calmodulin - dependent myosin light - chain kinase and 
  dephosphorylation by a type 1 phosphatase are the principal mechanisms of 
  regulation of smooth muscle tone 
  ( 35 ) .
given this evidence , it 
  is possible that phosphorylation of mtip and/or gap45 will have consequences 
  on the force generated by myoa .
apart from the potential role of gap45 phosphorylation in an interaction 
  with mtip or in motor control as suggested above , are there further 
  possibilities ?
gap45 has only been detected in apicomplexan parasites , and 
  apart from the n - terminal acylation sites and a 50-amino acid c - terminal 
  domain , the protein is poorly conserved and of low complexity composition .
it 
  is possible that phosphorylation confers structure by adding additional 
  negative charge to this low complexity region or facilitates interaction with 
  other molecules important in motor function , such as gap50 or others that have 
  yet to be identified .
we have shown that k252a can inhibit cdpk1 in vitro , with an 
  ic50 of 45 nm .
treatment of late stage p. 
  falciparum cultures with k252a also inhibited invasion of erythrocytes by 
  merozoites , but with a much higher ic50 of 348 nm . at 
  high concentrations of k252a
the egress of merozoites from schizonts was 
  inhibited , whereas at intermediate concentrations egress was not affected , but 
  merozoites failed to invade erythrocytes at the same rate as solvent - treated 
  control parasites .
the two distinct effects of k252a on parasite development 
  could be due to non - specific inhibition of parasite kinases at high 
  concentrations of inhibitor and a more specific inhibition of cdpk1 at 
  intermediate concentrations .
however , we can not exclude that there are other 
  cdpk1 substrates that play a crucial role in merozoite egress from schizonts 
  or that gap45 and mtip are important in this process , as well as their role in 
  invasion . while this paper was under review kato et al . 
 
( 36 ) published an article in 
  which ontology - based pattern identification was used to establish potential 
  targets of cdpk1 . in this
analysis proteins of the motor complex , including 
  mtip and gap45 , were identified as possible substrates .
the authors identified 
  a 2,4,6-trisubstituted purine compound , purfalcamine , that inhibits cdpk1 
  activity in vitro and inhibits merozoite egress from schizonts in 
  culture .
it is interesting that , like treatment with high concentrations of 
  k252a in our study , treatment of parasites with purfalcamine also results in 
  an inhibition of merozoite egress from the schizont .
previous investigations 
  have demonstrated that parasite kinase activity is important for invasion of 
  erythrocytes by merozoites .
treatment of parasites with the broad spectrum 
  kinase inhibitor staurosporine resulted in merozoites arrested on the surface 
  of erythrocytes , after the formation of a junction between the two cells 
  ( 37 ) .
in addition , the 
  importance of calcium in invasion and parasite motility has been shown in 
  several studies 
  ( 38 - 40 ) . 
 
phosphorylation of motor complex components by a calcium - dependent kinase may 
  therefore be a crucial regulator of motility and host cell invasion by 
  plasmodium .
 calcium - dependent protein kinases ( cdpks ) of apicomplexan 
  parasites are crucial for the survival of the parasite throughout its life 
  cycle . cdpk1 is expressed in the asexual blood stages of the parasite , 
  particularly late stage schizonts . 
 we have identified two substrates of 
  plasmodium falciparum cdpk1 : myosin a tail domain - interacting protein 
  ( mtip ) and glideosome - associated protein 45 ( gap45 ) , both of which are 
  components of the motor complex that generates the force required by the 
  parasite to actively invade host cells . 
 indirect immunofluorescence shows that 
  cdpk1 localizes to the periphery of p. falciparum merozoites and is 
  therefore suitably located to act on mtip and gap45 at the inner membrane 
  complex . 
 a proportion of both gap45 and mtip is phosphorylated in schizonts , 
  and we demonstrate that both proteins can be efficiently phosphorylated by 
  cdpk1 in vitro . 
 a primary phosphorylation of mtip occurs at serine 
  47 , whereas gap45 is phosphorylated at two sites , one of which could also be 
  detected in phosphopeptides purified from parasite lysates . 
 both cdpk1 
  activity and host cell invasion can be inhibited by the kinase inhibitor 
  k252a , suggesting that cdpk1 is a suitable target for antimalarial drug 
  development . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION