1. Paper Introduction
Kazuya Matsuo

2. Associate Professor @ Wayne State University
Angew. Chem. Int. Ed. 2015, 54, 9618–9621.
Angew. Chem. 2015, 127, 9754–9757.
① HDAC (= Histon Deacetylase) inhibitors for epigenetics
Mary Kay H. Pflum
Associate Wayne State University
From her homepage (
② Functional ATP (= Adenosine Triphosphate) analogue for kinase-catalyzed modification
From her homepage (
Kinase’s co-substrate is ATP

3. Previous works on kinase-catalyzed labeling
kinase substrate (protein)
kinase product (protein)
Pflum, M. K. H. et al. J. Am. Chem. Soc. 2007, 129,
Pflum, M. K. H. et al. ACIE 2010, 49,
ATP-biotin
Streptavidin (SA) has high affinity with biotin (Kd = mol/L)
All of these excellent works were done only in vitro because of cell-impermeability of ATP analogue

4. Basic information on cell membrane permeability
outside
inside
In passive transport
Relatively hydrophobic
Not high molecular weight (preferably < 500)
Positive charge > Negative charge although neutral is the best
※ Active transport (protein-mediated transportation using ATP) is not included
H2O
permeable
Noncharged molecules
Peptide
ATP
ion
Macromolecule
Impermeable
Cell membrane is a big barrier for ATP and its analogue because of their high negative charge
Novel ATP analogue for in-cell study
← APB (2)
To avoid side reaction, they used not 2º amine but 3º amine
Cationic linker was used in APB

5. Docking simulation of APB and in vitro kinase-catalyzed modification
Basic information of kinase
• >500 kinases and their thousands of substrates are identified so far
• Phosphorylation using ATP
• Regulation of metabolic and cell signaling
• Disorder of kinases can induce Parkinson’s disease and cancer
kinase
(ATP)
phosphatase
Substrate
of kinase
Product
of kinase
Docking model of PKA (kinase) with APB
Biotin labeling
Phosphorylation
※ 50% TFA treatment makes phosphate ester to hydrolyze
1. Biotin group is positioned outside active pocket
2. Catalytic Lys is located around phosphate groups
MBP : Substrate protein of PKA
PKA reacted with APB in vitro (APB = co-substrate of PKA)

6. In cellulo kinase-catalyzed modification
In vitro (HeLa cells lysate) labeling
Fluorescence microscopy analysis
※ HI lysate means heat-denatured lysate including no active kinase
After fixation of cells, biotinylated proteins were visualization by SA-Cy5
※ NHS-biotin show the random labeling of the proteins on cell surface
※ Fixation of cells = Method for imaging and observing cells
Fixed cells are not live cells, and the cell membrane is destroyed, so anti-body or fluorescent proteins can easily go into cells.
In vitro labeling showed selective biotinylation of kinase substrate using ATP-biotin and APB (this study)
Only APB can penetrate cell membrane
In cell labeling
※ STSP (staurosporine) : kinase inhibitor
In cell biotinylation by endogenous kinase was observed using APB
Labeling patterns between lysate (lane 4) and in cell (lane 11) are different
In cell labeling results show the real phosphoproteome much better
Kinase-catalyzed biotinylation using APB inside cells was achieved, so APB can go through cell membrane.
APB is the first cell-permeable ATP analogue.

7. Conclusion
Cell membrane
outside
inside
substrate
kinase
APB
product
biotinylation
APB is cell-permeable analogue of ATP, which can modify the kinase-substrate inside cells

8. From Wada-san notebook
Questions
According to Wada-san’s results and this paper, kinesin does not hydrolyze ATP-Az, but ATP-Az might be applicable to kinase
Is ATP recognition in kinesin and kinase completely different ?
ATP-Az
From Wada-san notebook
In both of kinesin and kinase,
① Mg ion activates the electrophilicity of ATP ② Leaving group = ADP
Kinesin → Nucleophile = water molecule (activated by kinesin)
Kinase → Nucleophile = hydroxyl group (activated by kinase)

9. I’ll try... ATP-Az Azo-TP caged ATP
Structure and activity relationship of γ-phosphate substituted ATP was well-studied
Shorter linker of ATP analogue is not preferable in kinase-catalyzed labeling reaction
Is ATP-Az with longer linker applicable to kinase or kinesin ?
Is this linker length important to show ATPase activity ?
ATP-Az
Azo-TP
Caged ATP before photolysis has no affinity with kinesin and kinase
caged ATP
Can kinase recognize Azo-TP ?
If possible, is Azo-TP photo-controllable for kinase-catalyzed phosphorylation ?
I’ll try...
Pflum, M. K. H. et al. Bioconjugate Chem. 2012, 23, 2386−2391.