Phospho-regulation of human Protein Kinase Aurora-A by K2Tytb9

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									                                     Supplementary Data
Fig. S1. Characterization of bacterially expressed recombinant Aurora-A.
We initially characterized bacterially expressed recombinant Aurora-A (rAurora-A) to
determine its biochemical properties. First, the kinase activity of rAurora-A was obviously
reduced after exposure to phosphatase but not in the presence of sodium vanadate
(Supplementary Figure 1a, lower panel). Second, targeted-mode mass spectrometry analysis
discovered that a segment of rAurora-A harbored the phosphorylated residue,
286
      RTTLCGTLDYLPPEMIEGR304 and that a phosphate group was located at Thr288, not
Thr287 from observations of fragment ion derivatives (Supplementary Figure 1b). These
findings were in agreement with those of others who identified this site as being involved in
regulating kinase activity in vivo (Walter et al., 2000). Finally, TPX2 is a physiological
activator of Aurora-A (Bayliss et al., 2003; Eyers et al., 2003; Eyers and Maller, 2004; Tsai
et al., 2003). rAurora-A with GST-TPX2(1-43) was 8-fold more active than rAurora-A with
the GST moiety alone on substrate GST-H3(5-15) (Supplementary Figure 1c). These results
revealed that rAurora-A was enzymatically and structurally active and that the kinase activity
depended on its phosphorylation at Thr288 in the activation loop, consistent with the
previous observations (Bayliss et al., 2003).
(A) Recombinant Aurora-A is phosphorylated, and phosphorylation is required for Aurora-A
activity.   His6-tagged   Aurora-A    was   expressed   in   E.   coli   and   purified   using
Ni2+-nitrilotriacetic acid agarose. Recombinant Aurora-A was incubated for 30 min at 30˚C
with 100 units of -PPase (New England Biolabs) in phosphatase buffer (50 mM Tris-HCl,
100 mM NaCl, 2 mM MnCl2, 2 mM dithiothreitol, 0.1 mM EGTA, 0.01 % Brij 35) with or
without 10 mM sodium orthovanadate (Sigma). Purified (lane 1), -PPase-treated (lane 2)
and sodium vanadate plus -PPase-treated rAurora-A (lane 3) were probed with anti-pan
Aurora antibody (K3-7) (upper panel) and used in kinase assays in vitro (IVK) with 10 mM
sodium orthovanadate and recombinant GST fusion protein containing histone H3 amino
terminus (residues 5-15) as substrate. Reaction products separated by SDS-PAGE, were
immunoblotted against anti-phospho-histone H3 (pSer10) antibody.
(B) Tandem mass spectrum of a tryptic peptide from the activation loop of rAurora-A,
showed singly phosphorylated amino acid residue Thr288A. We digested rAurora-A with
modified trypsin (Promega) in 50 mM NH4HCO3 at 37˚C for 8 h. The reaction was stopped
by adding a final concentration of 0.1% formic acid. The resulting peptide mixture was
analyzed by liquid chromatography/mass spectrometry (LC/MS) and MS/MS. Mass
spectrometry proceeded using a hybrid Q-TOF instrument (Micromass). Tandem MS
sequencing was performed by selection and fragmentation of the precursor ion with an m/z
value corresponding to the [M+2H]2+ ion of the trypsin fragment. Protein was identified using

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the commercially available search engine Mascot (Matrixscience). Interpretation of the
MS/MS spectrum ([M+2H]2+ = m/z 1123) revealed conclusive b/y-type fragment ions that
defined phosphorylation on Thr288.
(C) GST-TPX2 (1–43) increases kinase activity of rAurora-A. In vitro phosphorylation
(32P-ATP) of GST-H3 (5-15) by rAurora-A in the presence of GST (lane 1) or GST-TPX2
(1–43) (lane 2), or GST-TPX2 (1–43) alone (lane 3). Upper panel, autoradiogram of
SDS-PAGE gel; middle panel, corresponding Coomassie blue-stained gel. Equal amounts of
rAurora-A were confirmed by staining with Coomassie blue (lower panel). Notably,
GST-TPX2(1-43) was efficiently phosphorylated (lane 2), a fact that is addressed in detail in
the Discussion section.


Referrence:
Bayliss R, Sardon T, Vernos I and Conti E. (2003). Structural basis of Aurora-A activation by
         TPX2 at the mitotic spindle. Mol Cell 12: 851-862.
Eyers PA, Erikson E, Chen LG and Maller JL. (2003). A novel mechanism for activation of
         the protein kinase Aurora A. Curr Biol 13: 691-697.
Eyers PA and Maller JL. (2004). Regulation of xenopus aurora A activation by TPX2. J Biol
         Chem 279: 9008-9015.
Tsai MY, Wiese C, Cao K, Martin O, Donovan P, Ruderman J, Prigent C and Zheng Y.
         (2003). A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle
         assembly. Nat Cell Biol 5: 242-248.
Walter AO, Seghezzi W, Korver W, Sheung J and Lees E. (2000). The mitotic
         serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and
         degradation. Oncogene 19: 4906-4916.



Fig. S2. Mutations in activation loop of Aurora-A affect kinase activity.
We reconfirmed the kinase activity by repeating the in vitro kinase assay using the
glu-Aurora-A T288A mutant immunoprecipitated from mammalian cells and revealed that
mutant T288A caused the substantially defective autophosphorylation and phosphorylation of
GST-H3(5-15). Wild-type or Thr288 mutants of glu-Aurora-A were transiently expressed in
COS cells under conditions indicated at top of panels. In vitro kinase assay of glu-Aurora-A
immunoprecipitates (upper panel) and Coomassie blue staining in middle and lower panels
shows total amounts of kinases and substrate, respectively. Thr288 mutants of glu-Aurora-A
caused     the   substantially   defective   autophosphorylation   and   phosphorylation   of
GST-H3(5-15). These data also excluded the notion that a contaminating kinase(s) might

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have co-purified with glu-Aurora-A and also caused phosphorylation in the assay.


Fig. S3. Specificity of the phospho-Thr288 Aurora-A-specific monoclonal
antibody pA7.2.
Three independent lines of evidence testified to the specificity of the monoclonal
phospho-specific antibody pA7.2. Firstly, the antibody selectively recognized the
phosphorylated peptide in ELISA screening assays but not the opposing non-phosphorylated
peptide (data not shown). Secondly, we used peptide competition by incubating the antibody
before application to the immunoblot membrane, with phosphorylated or non-phosphorylated
peptide. Although blocking the antibody with unphosphorylated peptide had no effect,
blocking with phosphorylated peptide abolished the signal (data not shown). Thirdly, the
antibody immunoprecipitated the slower of the two electrophoretic forms of Aurora-A from
transfected COS cell lysates (Supplementary Figure 3a, lanes 2 and 7). Neither human
Aurora-B nor Aurora-C was immunoprecipitated with the pA7.2 antibody, showing that not
only the phosphorylation of threonine but also the amino acid sequence surrounding the
phosphorylated threonine residue is a requirement. The pA7.2 antibody immunoprecipitated
faint amounts of a catalytically inactive mutant (KN) of Aurora-A (Supplementary Figure 3a,
lane 8). This result validated the notion of an upstream kinase(s) that phosphorylates Thr288
of Aurora-A in vivo. However our data do not exclude the possibility that endogenous
Aurora-A or residual kinase activity of the mutant is responsible for Thr288 phosphorylation.
        To assess the ability of the monoclonal anti-phospho-Aurora-A antibody pA7.2 to
detect endogenous proteins, HeLa cells were incubated in the presence or absence of
nocodazole, and then cell lysates were immunoprecipitated with the antibody. Supplementary
Figure 3b, lanes 1 and 2 show that nocodazole increased the protein level of Aurora-A as
cells accumulated in prometaphase. A major band and an apparent mobility shifted band of
Aurora-A from lysates of nocodazole-treated cells were immunoprecipitated with anti-pan
Aurora monoclonal antibody (Supplementary Figure 3b, lane 4). The pA7.2 antibody
specifically immunoprecipitated the slowly migrating band (Supplementary Figure 3b, lane
6), confirming its ability to detect endogenous and phosphorylated Aurora-A.
(A) The phospho-Thr288 Aurora-A-specific monoclonal antibody pA7.2 immunoprecipitated
the slower of the two electrophoretic forms of Aurora-A. Neither human Aurora-B nor
Aurora-C was immunoprecipitated with the pA7.2 antibody. Three subfamilies of human
Aurora (glu-tagged) or glu-Aurora-A (KN) were transiently expressed in COS cells.
Immunoblots of total cellular lysates (left panel) and pA7.2 immunoprecipitates (right panel)
were probed with anti-glu antibody. Only the slowly migrating electrophoretic form of


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Aurora-A was immunoprecipitated by pA7.2 antibody from transfected COS cell lysates
(lanes 2 and 7). Antibody immunoprecipitated faint amounts of a catalytically inactive mutant
(KN) of Aurora-A (lane 8). Asterisk represents heavy chains of mouse monoclonal pA7.2
antibody used for immunoprecipitation.
(B)   Only   the   more     slowly   migrating       species   of   endogenous   Aurora-A   was
immunoprecipitated by pA7.2. HeLa cells were either grown exponentially or incubated with
400 ng/ml of nocodazole (Noco) for 8 h to induce arrest at mitosis. Lysates of HeLa cells
were immunoprecipitated with anti-pan Aurora K3-7 (lanes 3 and 4) or anti-pA 7.2 antibodies
(lanes 5 and 6), respectively. A major band and an apparent mobility shifted band of
Aurora-A from lysates of nocodazole-treated cells were immunoprecipitated with anti-pan
Aurora monoclonal antibody (lane 4). The pA7.2 antibody specifically immunoprecipitated
the slowly migrating band (lane 6), confirming its ability to detect endogenous and
phosphorylated Aurora-A. Asterisk indicates heavy chains of mouse monoclonal antibodies
used for immunoprecipitation.



Fig. S4. Aurora-A forms complexes with PP1.
(A) Alignment of consensus PP1 binding motif and corresponding Aurora-A sequences.
Kinase domain of Aurora-A contains two predicted sequences KVLF (at position 162-165)
and RVEF (at position 343-346).
(B) Aurora-A interacts with endogenous PP1 in vivo. GST or GST-Aurora-A expressed in
COS cells were pulled down using glutathione-Sepharose beads, and total lysates and
complexes (AP) were assayed by immunoblotting for anti-PP1 (upper panel) or anti-GST
(lower panel) antibodies.
(C) Aurora-A bound three isoforms of catalytic subunit of human PP1. Isoforms of catalytic
subunit of human PP1 (myc-tagged) or glu-Aurora-A were transiently expressed either alone
or together in COS cells under transfection conditions indicated at top of panels.
Immunoblots of glu-Aurora-A immunoprecipitates (upper) and total cell lysates (lower) were
probed with anti-glu and anti-myc antibodies. Positions of molecular mass standards are
indicated on left of each panel. Asterisks represent heavy chains of mouse monoclonal
anti-glu antibody used for immunoprecipitation.
(D) Aurora-A mutated at predicted motifs bound PP1. Hydrophobic and aromatic residues
(Val163-L-Phe165 and Val344-E-Phe346) conserved in this motif were mutated to alanine, either
independently or together (FA mutant I; Ala163-L-Ala165, II; A344-E-A346, and I + II). COS
cells were transfected with cDNA encoding wild-type or mutated glu-Aurora-A together with
myc-tagged catalytic subunits of PP1 or empty vector. Immunoblots of glu-Aurora-A

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immunoprecipitates were probed with anti-glu and anti-myc antibodies. Asterisks represent
heavy chains of mouse monoclonal anti-glu antibody used for immunoprecipitation.
(E) Aurora-A associated with PP1 in nocodazole-treated cells. Complementary DNAs
encoding glu-Aurora-A and empty vector or myc-PP1 were transiently expressed in COS
cells. Transfected cells were incubated with 400 ng/ml of nocodazole (Noco) for 8 h before
lysis. Immunoblots of glu-Aurora-A immunoprecipitates (upper) and total cellular lysates
(lower) were probed with anti-glu and anti-myc antibodies. Asterisks represent heavy chains
of mouse monoclonal anti-glu antibody used for immunoprecipitation.




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