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18 Investigation<br />
Section III: Workflow Tools<br />
using motif and post-translational<br />
modification-specific antibodies<br />
Motif and post-translational modification (PTM) antibodies<br />
can help answer two major scientific questions.<br />
1. How can I measure global changes in PTMs or protein kinase activity in response<br />
to cellular treatments<br />
2. How can I investigate PTMs on a protein of interest without complex in vivo labeling<br />
experiments or where no target-specific antibody is available<br />
Post-translational modifications of proteins catalyzed by kinases, acetylases, methylases, ubiquitin<br />
ligases, and other enzymes are key regulators of cell signaling and can be valuable targets for therapeutic<br />
intervention in disease states. Antibodies recognizing specific protein modification sites (e.g.<br />
Phospho-Akt, Acetyl-Histone) are well-known and valuable tools for studying PTMs. To augment these<br />
tools, <strong>CST</strong> has developed motif and PTM-specific antibodies, a distinct class of PTM reagents that<br />
have a broader target specificity and can therefore be used for a different set of applications.<br />
Motif Antibodies<br />
PTMs catalyzed by a specific modification enzyme are often constrained to a peptide sequence pattern,<br />
or motif, that reflects the substrate specificity of that enzyme. For example, substrates of the<br />
protein kinase Akt generally contain an RXRXXS motif in which Akt phosphorylates the serine residue.<br />
Antibodies that detect the motif RXRXXpS can be used to detect a broad spectrum of Akt substrates.<br />
Motif antibodies, proprietary to <strong>CST</strong>, recognize a modified residue in the context of a specific peptide<br />
sequence motif. These highly specialized antibodies were originally developed for the identification<br />
and quantification of PTMs catalyzed by kinases from several branches of the human kinome. More<br />
recently, individual motif antibodies have been developed to recognize several other post-translational<br />
modifications such as methyl-arginine and caspase cleavage sites.<br />
PTM-specific Antibodies<br />
PTM-specific antibodies differ from motif antibodies in that they recognize a specific protein posttranslational<br />
modification, but are not restricted to a specific sequence motif surrounding the modification<br />
site. Examples of PTM-specific antibodies from <strong>CST</strong> include Phospho-Tyrosine (P-Tyr-1000) Rabbit<br />
mAb #8954 and Acetylated-Lysine (Ac-K 2 -100) Rabbit mAb #9814. Together, motif and PTM-specific<br />
antibodies provide a robust toolbox for the identification and classification of PTMs and their effects on<br />
cellular regulation.<br />
How can motif and PTM-specific antibodies be used<br />
• Quantitatively identify the PTM alterations of cellular proteins due to up- or down-regulation<br />
of a specific kinase, acetylase, methylase, nitrosylase, or ubiquitin ligase.<br />
• Motif and PTM antibodies can be used in early-stage investigations where a high quality<br />
antibody may not be available for a specific modified protein target. Assay target-specific<br />
PTM alterations via immunoprecipitation (IP) enrichment of the target protein followed by<br />
western blot (WB) analysis using the motif or PTM-specific antibody.<br />
• Screen candidate therapeutics using in vitro kinase and phosphatase assays based on a<br />
motif or PTM-specific antibody.<br />
One example of the application of a motif antibody involves the response of Jurkat cells to the PP1 and<br />
PP2A protein phosphatase inhibitor Calyculin A. The response was studied by 2D gel electrophoresis<br />
followed by WB analysis using a <strong>CST</strong> motif antibody recognizing the phospho-serine 14-3-3 binding<br />
motif. The data reveal a pronounced increase in overall phosphorylation levels. Based on this initial<br />
study, individual proteins of interest are selected for further study.<br />
Specific kinases in the human kinome phosphorylate<br />
target proteins at distinct substrate sequence motifs.<br />
CHED<br />
Haspin<br />
STLK3<br />
MAST4<br />
chapter 18: Investigation<br />
TK<br />
FGFR2 FGFR3<br />
TrkC<br />
EphB2<br />
TrkB<br />
FGFR1<br />
FGFR4<br />
EphB1<br />
TrkA<br />
FLT1/VEGFR1<br />
ATM<br />
EphA5<br />
KDR/VEGFR2<br />
MuSK ROR2<br />
Fms/CSFR<br />
Atypical<br />
ROR1<br />
EphB3<br />
Ret<br />
Kit<br />
EphA3<br />
DDR2<br />
Mer<br />
ATM<br />
EphA4<br />
DDR1<br />
Tyro3/<br />
Axl<br />
FLT4<br />
Sky<br />
ATR<br />
PDGFRα<br />
EphA6<br />
IGF1R IRR<br />
FLT3<br />
PDGFRβ<br />
mTOR/FRAP<br />
InsR<br />
Yes<br />
EphB4<br />
Met<br />
EGFR<br />
PIKK<br />
HER2/ErbB2<br />
Ron<br />
DNAPK<br />
Src<br />
MLK3<br />
Ros<br />
SMG1<br />
EphA7<br />
ALK<br />
MLK1<br />
Lyn<br />
LTK<br />
Tie2<br />
Tie1<br />
TRRAP<br />
HCK<br />
Fyn<br />
RYK<br />
HER4<br />
EphA8<br />
MLK4<br />
CCK4/PTK7<br />
TKL<br />
MLK2<br />
Lck<br />
Fgr<br />
Ack Tnk1 Tyk2<br />
Jak1<br />
HER3<br />
Jak2<br />
EphA2<br />
Jak3<br />
BLK<br />
ANKRD3 SgK288<br />
DLK<br />
Syk Zap70/SRK<br />
EphA1<br />
PYK2/FAK2<br />
LZK<br />
FAK<br />
Lmr1<br />
ALK4<br />
ITK<br />
Lmr2<br />
C-Raf/Raf1<br />
FRK<br />
TGFβR1<br />
ZAK<br />
BRaf<br />
TEC<br />
EphB6<br />
KSR<br />
Srm<br />
RIPK2<br />
KSR2<br />
TXK<br />
Brk<br />
BTK<br />
Lmr3<br />
ALK7<br />
IRAK3<br />
IRAK1<br />
LIMK1<br />
ARaf<br />
BMPR1B<br />
Etk/BMX<br />
EphA10<br />
LIMK2 TESK1 ILK<br />
BMPR1A<br />
CTK<br />
RIPK3<br />
TSK2<br />
TAK1<br />
HH498<br />
ALK1<br />
CSK CK2a1<br />
PAK1<br />
ALK2<br />
Abl2/Arg<br />
IRAK2<br />
ActR2<br />
Abl Fes<br />
ActR2B<br />
STE<br />
Fer<br />
RIPK1<br />
Jak3~b<br />
TGFβR2<br />
LRRK2<br />
MEKK2/MAP3K2<br />
Jak2~b<br />
LRRK1<br />
MISR2<br />
MEKK3/MAP3K3<br />
Tyk2~b<br />
SuRTK106<br />
IRAK4<br />
BMPR2<br />
ASK/MAP3K5<br />
ANPα/NPR1<br />
MAP3K8<br />
Jak1~b<br />
MAP3K7<br />
ANPβ/NPR2<br />
KHS1<br />
MOS<br />
KHS2<br />
HSER<br />
SgK496<br />
WNK1<br />
WNK3<br />
MEKK6/MAP3K6<br />
DYRK2<br />
Mst4<br />
PBK<br />
MAP3K4<br />
DYRK3<br />
GUCY2D<br />
WNK2<br />
DYRK4<br />
DYRK1A<br />
NRBP1<br />
GUCY2F<br />
NRBP2 MEKK1/MAP3K1 OSR1<br />
DYRK1B<br />
WNK4<br />
MLKL<br />
PERK/PEK<br />
SgK307<br />
SLK<br />
PKR<br />
LOK<br />
HIPK1 HIPK3<br />
GCN2 SgK424<br />
TAO1<br />
SCYL3 TAO2<br />
HIPK2<br />
SCYL1<br />
Tpl2/COT<br />
SCYL2<br />
NIK<br />
TAO3<br />
PAK1<br />
CLK4 HIPK4<br />
PRP4<br />
HRI<br />
PAK3<br />
CLIK1<br />
PAK2<br />
IRE1<br />
CLK2 CLK1<br />
PAK4<br />
MAP2K5<br />
PAK5/PAK7<br />
CLIK1L<br />
IRE2<br />
CLK3<br />
TBCK<br />
PAK6<br />
MAP2K7<br />
MEK1/MAP2K1<br />
RNAseL<br />
GCN2~b<br />
MEK2/MAP2K2<br />
TTK<br />
MSSK1<br />
SgK071<br />
KIS<br />
GRK2<br />
CMGC<br />
SRPK2<br />
MYT1<br />
SEK1/MAP2K4 MKK3/MKK6<br />
SRPK1<br />
CK2α1<br />
Wee1<br />
MAK<br />
CK2α2<br />
SgK196<br />
CDC7<br />
Wee1B<br />
CK1δ<br />
ICK<br />
PRPK<br />
TTBK1<br />
CK1ε<br />
GSK3β<br />
MOK<br />
TTBK2<br />
GSK3α<br />
CK1α1<br />
CDKL3<br />
CK1α2<br />
CDKL2<br />
PINK1<br />
SgK493<br />
SgK269<br />
VRK3<br />
CK1γ2<br />
CDKL1 CDKL5<br />
ERK7<br />
SgK396<br />
SgK223<br />
CDKL4 Erk4<br />
Slob<br />
CK1γ1<br />
Erk3<br />
SgK110<br />
PIK3R4<br />
CK1γ3<br />
NLK<br />
SgK069<br />
Bub1<br />
Erk5<br />
SBK<br />
BubR1<br />
Erk1/<br />
IKKα<br />
p44MAPK<br />
IKKβ<br />
VRK1<br />
CDK7<br />
IKKε<br />
VRK2<br />
Erk2/<br />
Erk2<br />
CK1<br />
PLK4<br />
PITSLRE<br />
TBK1/NAK<br />
p42MAPK p38γ<br />
MPSK1<br />
JNK1<br />
p38δ<br />
JNK2<br />
TLK2<br />
JNK3 CDK10<br />
GAK<br />
TLK1<br />
PLK3<br />
p38β<br />
AAK1<br />
CDK8 CDK11<br />
CAMKK1<br />
ULK3<br />
PLK1<br />
p38α<br />
PLK2<br />
CDK4<br />
CCRK<br />
BIKE<br />
CAMKK2<br />
BARK1/GRK2<br />
CDK6<br />
ULK1<br />
BARK2/GRK3 RHOK/GRK1<br />
Fused<br />
GRK5<br />
PFTAIRE2<br />
SgK494<br />
ULK2 ULK4<br />
GRK6 GRK4<br />
PFTAIRE1<br />
CDK9<br />
Nek6<br />
RSKL1<br />
PCTAIRE2<br />
Nek7 Nek10<br />
SgK495<br />
PASK<br />
PDK1<br />
RSKL2<br />
Nek8<br />
MSK1<br />
RSK1/p90RSK<br />
PCTAIRE1<br />
CDK5 CRK7<br />
PCTAIRE3<br />
Nek9<br />
LKB1<br />
MSK2<br />
RSK4 RSK2<br />
Chk1<br />
p70S6K<br />
RSK3<br />
Nek2<br />
Akt2/PKBβ<br />
AurA/Aur2<br />
p70S6Kβ<br />
Akt1/PKBα<br />
cdc2/CDK1<br />
Nek11<br />
CDK3<br />
CDK2<br />
Nek4<br />
AurB/Aur1<br />
Trb3 Pim1<br />
AurC/Aur3<br />
Pim2<br />
LATS1<br />
Nek3<br />
Pim1Trb2<br />
Pim3<br />
Nek5<br />
Trad Trio<br />
LATS2<br />
NDR1<br />
Trb1<br />
Obscn~b<br />
NDR2<br />
Nek1<br />
YANK1<br />
SPEG~b<br />
MAST3<br />
MASTL<br />
Obscn<br />
STK33<br />
YANK2 PKN1<br />
YANK3<br />
SPEG<br />
TTN<br />
MAST2<br />
SgK085<br />
caMLCK<br />
MAPKAPK2<br />
skMLCK<br />
smMLCK<br />
DRAK2<br />
DRAK1<br />
DAPK2<br />
DAPK3<br />
DAPK1<br />
SSTK<br />
TSSK3<br />
TSSK1<br />
TSSK2<br />
AMPKα2<br />
AMPKα1<br />
BRSK2<br />
CAMK<br />
BRSK1<br />
SNARK<br />
ARK5<br />
QSK<br />
SNRK<br />
NIM1<br />
SIK<br />
QIK<br />
TSSK4<br />
MELK<br />
MARK4<br />
MARK3<br />
MARK1<br />
MARK2<br />
HUNK<br />
PKD2/PKCµ<br />
PKD1<br />
PKD3/PKCν<br />
MNK1<br />
MNK2<br />
RSK4~b<br />
RSK1~b<br />
RSK2~b<br />
RSK3~b<br />
CASK<br />
MAPKAPK5<br />
MAPKAPK2<br />
MAPKAPK3<br />
MSK2~b<br />
MSK1~b<br />
Chk2/Rad53<br />
STRAD/STLK5<br />
STLK6<br />
CaMKIβ<br />
CaMKIγ<br />
MAST1<br />
DCAMKL3<br />
DCAMKL1<br />
DCAMKL2<br />
VACAMKL<br />
PhKγ1<br />
PhKγ2<br />
PSKH1<br />
CaMKIIγ<br />
PSKH2<br />
CaMKIIα<br />
CaMKIIβ<br />
CaMKIIδ<br />
CaMKIV<br />
CaMKIα<br />
CaMKIδ<br />
GRK7<br />
HPK1<br />
GCK<br />
Akt3/PKBγ<br />
SGK1<br />
SGK2<br />
SGK3<br />
PKG2<br />
PKN1/PRK1<br />
PKG1<br />
PKN2/PRK2<br />
PKN3<br />
PRKY<br />
PKCδ<br />
PKCθ<br />
PRKX<br />
PKCη<br />
PKCε<br />
PKCι<br />
PKCζ<br />
PKAγ<br />
PKAα<br />
PKCγ<br />
PKAβ<br />
AGC<br />
ROCK1<br />
PKCα<br />
ROCK2<br />
PKCβ<br />
DMPK<br />
CRIK<br />
DMPK2<br />
MRCKβ<br />
MRCKα<br />
Representative phosphorylation motifs from the PhosphoSitePlus ® online resource. Logos for individual motifs are superimposed on the kinase dendrogram developed in collaboration<br />
between Gerard Manning and Cell Signaling Technology (1). These and over 100 other kinase motifs can be generated for kinases that have 15 or more unique protein substrates reported<br />
in the literature, and these are curated into the PhosphoSitePlus ® knowledge base (2).<br />
References<br />
1. Manning, G., et al. (2002) Science 298, 1912–1934.<br />
2. Hornbeck, P.V., et al. (2012) Nucleic Acids Res. 40, 261–270.<br />
NRK/ZC4<br />
Mst1<br />
Mst2<br />
TNIK/ZC2<br />
MYO3A<br />
MYO3B<br />
YSK1<br />
Mst3<br />
HGK/ZC1<br />
MINK/ZC3<br />
Akt<br />
254 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.<br />
www.cellsignal.com/investigation<br />
255
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