1. A cell-permeable ATP analog for kinase-catalyzed biotinylation
Ahmed E. Fouda and Mary Kay H. Pflum
Wayne State University, Department of Chemistry, Detroit, MI, 48202
ABSTRACT 5 9
Synthesis of ATP-polyamine-biotin (APB)
Cell permeability of APB
ATP-polyamine-biotin (APB, 2) was synthesized from commercially available spermine (3; Scheme 2). Spermine (3) was protected at the primary amine with Boc-ON, followed by reductive amination at the secondary amine to give compound (5). 5 was deprotected with 4M HCl to give methyl spermine 6. NHS-ester of biotin was used to convert methyl spermine (6) into polyamine biotin (7). Finally, polyamine biotin (7) was coupled with ATP in presence of EDCI at pH= to get ATP polyamines biotin (2).
To test the cell permeability of APB, direct fluorescence microscopy was used. APB was incubated with Hela cells, followed by washing, fixing, and permeabilizing the cells, then incubated with streptavidin-Cy5 conjugate and scanned with fluorescence microscope. Cells treated with APB showed fluorescence corresponding to biotin, which confirms the permeability of APB [Figure 8]. As a control, untreated cells or cells incubated with ATP or ATP-biotin showed no fluorescence corresponding to biotin [Figure 8]. The microscopy experiment suggests that the polyamine linker in APB is required to promote cell permeability.
Kinase-catalyzed protein phosphorylation is a regulatory process controlling cascades of biochemical reactions. Irregularities in phosphorylation result in many diseases, such as diabetes mellitus, Parkinsons, and cancer. Therefore, development of new methods to monitor kinase-catalyzed phosphorylation is needed to understand normal and diseased states. The Pflum lab recently developed kinase-catalyzed biotinylation using ATP-biotin to monitor kinase enzymes and substrate phosphorylation. However, kinase-catalyzed biotinylation is limited to in vitro applications only due to cell impermeability of ATP analog. Here, we report the first cell permeable ATP analog, ATP-polyamine-biotin (APB), compatible with kinase-catalyzed biotinylation. APB showed in vitro protein labeling similar to ATP-biotin. APB was visualized in cell by fluorescence microscopy. Importantly, biotin labeling of kinase substrates in living cells was also observed. APB will aid in monitoring kinase-catalyzed phosphorylation in living cells, which will enhance studies on phosphoprotein purification and analysis. More generally, APB provides a foundation for development of additional cell permeable ATP analogs for cell signaling research..
DAPI
SA-Cy5
Merge
Untreated
ATP
ATP- biotin
APB
INTRODUCTION
Scheme 2: Synthesis of ATP-polyamine-biotin (APB). 1
Kinase-catalyzed protein phosphorylation 6
Living cells regulate extracellular signals through a variety of biochemical reactions in signaling cascades. One of the ubiquitous signaling reactions is kinase-catalyzed protein phosphorylation, which is the transfer of phosphate group from ATP to hydroxyl containing amino acids serine, threonine and tyrosine [Figure 1]. Kinase-catalyzed protein phosphorylation regulates many metabolic and cell signaling pathways and the alteration of pathways involving kinases can lead to diseases, such as cancer, Parkinson’s and Diabetes mellitus. For example, elevated levels of kinases are associated with B-Raf mutation which is mutated in 2% of all cancer. Therefore, studying protein kinases and their substrates will afford a better understanding of many diseases and cell signaling pathways.
In vitro Mylein Basic Protein (MBP) labeling with APB and PKA
To test the compatibility of APB with protein kinases, APB was incubated with PKA kinase and full length protein substrate, myelin basic protein (MPB), followed by SDS page gel analysis. After transfer to PVDF membrane, biotinylated MPB was visualized using Strepatavidine Cy-5 conjugate [Figure 5] and revealed a kinase-dependent biotinylation [lane 4, Figure 5]. In addition, biotinylation disappeared upon acid cleavage of phosphoramidate bond [lane 5, Figure 5] and in presence of kinase inhibitor staursporin [lane 8, Figure 5] . Therefore, we concluded that APB is a kinase co-substrate.
Sypro Ruby
Streptavidin-Cy5
Figure 8 – Microscopy studies of ATP-biotin or APB cell permeability. HeLa cells were untreated or treated with ATP, ATP-biotin or APB before visualization with the nuclear stain DAPI or biotin stain streptavidin-Cy5 (SA-Cy5). 10 1 2 3 4 5 6 7 8
MBP +
PKA -
ATP
APB
50% TFA
STSP
In cell protein labeling using APB
To test the cell permeability of APB, APB was incubated with Hela cells in 12 well plates. After washing with cold DPBS, cells were lysed, and biotinylation was analyzed by SDS-PAGE gel electrophoresis, followed by transfering to PVDF membrane. Biotinylation was detected by streptavidin Cy-5 conjugate. The analysis revealed that ATP-biotin did not show any protein labeling [Figure 9A, lane 2] due to cell impermeability. On the other hand, APB showed protein labeling [Figure 9A, lane 4], which suggests that it is cell permeable. Pretreating cells with staursporine, kinase inhibitor, showed less labeling [Figure 9A, lane 3], which suggests that the labeling is kinase dependent.
Figure 1: Protein phosphorylation of A) Serine (R=H) and threonine (R=Me), or B) tyrosine.
Figure 5: Kinase-catalyzed biotinylation of Myelin basic protein (MBP) with ATP or APB, in presence or abscence of PKA kinase. TFA (50% final concentration) was added after biotinylation labeling (lane 5) to cleave the phosphormaidate bond. As a control the reaction was performed in presence of the kinase inhibitor staursporine (STSP). The labeling mixtures were separated by SDS-PAGE and visualized with SYPRO® Ruby total proteins stain (bottom), or streptavidin-Cy-5 (top). 2
SA-Cy5
SYPRO Ruby
Kinase cosubstrate promiscuity
Kinases utilize ATP as a cosubstrate to phosphorylate proteins [Scheme 1]. Recently, the Pflum lab discovered the ability of kinase enzymes to utilize γ-modified ATP analogs with large groups attached to the terminal phosphate , a phenomenon called kinase cosubstrate promiscuity [Scheme 1]. For example, Pflum lab used a γ-modified ATP analog called ATP-biotin. ATP-biotin was able to label peptides or proteins in vitro and proteins in cell lysates, which makes it a tool to study phosphoproteomics. 7
Efficiency of phosphorylation with APB
To investigate the efficiency of biotinylation, APB was incubated with MPB and PKA, followed by cleavage of the biotin with TFA. The reaction mixtures were analyzed by SDS page gel analysis and staining with phosphoprotein stain ProQ diamond phosphoprotein stain. Phosphorylation was quantified by ImageQuant 5.2 on ProQ diamond stained gel. APB showed 55% (±6) conversion while ATP-biotin showed 72% (±7) compared to ATP (Figure 6). Next, kinetic studies were performed by incubating APB or ATP with PKA and kemptide peptide substrate. APB showed a reduced kcat/KM (0.25 s-1µM-1) compared to ATP (0.52 s-1µM-1). The observed quantitative analysis confirms that APB can label kinase substrates efficiently, although less efficiently than ATP-biotin. This data is consistent with the docking studies which showed that the g-phosphate of APB is little far from the catalytic K168 compared to ATP-biotin. 1 2 3 4
Hela cells +
ATP biotin -
APB
STSP
SYPRO RUBY
ProQ diamond
55%
72%
100%
Figure 9: In cellulo kinase-catalyzed biotinylation with ATP-biotin or APB in HeLa cells. As a control, kinase inhibitor staurosporine (STSP) was pre-incubated with cells to prevent kinase catalysis (lane 3). Reaction mixtures were separated by SDS-PAGE and visualized with streptavidin-Cy5 (SA-Cy5, top gel) or SYPRO® Ruby total protein stain (bottom gels).
Scheme 1: Mechanism of protein phosphorylation with ATP or ATP biotin (1) or APB (2) and kinases. 1 2 3 4
MBP +
PKA
ATP -
ATP biotin
APB
CONCLUSIONS and future directions 3
Cell-permeable ATP analog for kinase-catalyzed labeling
Figure 6: Quantitative analysis of MPB phosphorylation in presence of PKA and incubated with either ATP (lane 2), ATP-biotin 1 (lane 3), or APB 2 (lane 4), then treated with TFA. The reaction mixtures were analyzed by SDS PAGE gel analysis, followed by staining with ProQ diamond phosphoprotein stain (top) or SYPRO® Ruby total protein stain (bottom).
Due to the cell impermeability of ATP analogs, ATP-biotin utilization was limited to in vitro experiments with cell lysates. However, a cell permeable ATP analog will be more physiologically relevant due to compartmentalization. To create cell permeable ATP analog, we focused on enhancing cell permeability by removing or neutralizing the negative charge of the triphosphate. This approach is supported by previous reports increasing the cell permeability of phosphate compounds such as bisphosphonates and phosphoinisitols. Building on this precedent, we replaced the PEG linker of ATP-biotin (1, Figure 2) with a polyamine linker (2, Figure 2). The polyamine linker will be positively charged under physiological conditions, which can neutralize the triphosphate negative charges and promote cell permeability. We call this compound ATP-polyamine-biotin (APB).
In conclusion, we report the first cell permeable ATP analog; ATP polyamine biotin. APB acted as a cosubstrate with protein kinases in vitro and in cellulo. Importantly, different biotinylated proteins were observed with in cellulo compared to lysate studies, which argue that labeling in cellulo will better reflect the cellular phosphoproteome.
Future directions: APB and kinase-catalyzed biotinylation can inspire for future work to identify and isolate phosphoproteins from cells, which will enhance cell signaling research. More generally, these studies establish a general strategy for development of other ATP analogs for live cell labeling studies. 8
To further analyze the compatibility of APB with kinases in cells, APB was incubated with Hela cell lystaes followed by SDS page gel analysis. After transfer to PVDF membrane, biotinylation was detected [lane 4, Figure 7]. Two controls were used to assess the dependence of biotinylation on kinases. The first was heat denatured lysates which showed less biotinylation due to inactivation of kinases [lane 3, Figure 7]. Second, we incubated biotinylated proteins with 50% TFA to test if the polyamines biotin is attached via phosphoramidate bond. Biotinylation was decreased by acid [lane 5, Figure 7], suggesting that phosphoramidate bond was cleaved. In additon, APB showed same biotinylation levels [lane 4, Figure 7] as ATP-biotin [lane 7, Figure 7]. Biotinylation of cell lysates by APB suggests that APB is compatible with cellular kinases.
In vitro Hela cell lysates labeling with APB
Figure 7 – Kinase catalyzed biotinylation with APB (2) ATP-biotin (1) with Hela cell lysates (HL) and heat denatured hela cell lysates (DHL). Acid (50% trifluoroacetic acid final concentration) was added after biotin labeling to cleave the biotin tag (lane 5). Reaction mixtures were separated by SDS-PAGE and visualized with streptavidin-Cy5 (SA-Cy5, top gel) or SYPRO® Ruby total protein stain (bottom gels).
SYPRO RUBY
SA-Cy5
REFERENCES
Figure 2: Chemical structure of ATP biotin (1) and ATP polyamines biotin (APB) (2).
Results and Discussion 4
Docking of ATP-polyamine-biotin (APB)
APB comptability with kiases was analyzed computationally by docking APB with the PKA kinase crystal structure. Similar PKA binding was observed with APB (Figure 4A), ATP-biotin (Figure 4B), and ATP (Figure 4C). The a-phosphate of APB, ATP-biotin, and ATP is 3.8, 3.7, and 3.2 Å from the catalytic amino acid K72 respectively (Figure 4). In contrast, the g-phosphate of APB is 3.9 Å from K168 (Figure 4A), while it is 2.4 Å with both ATP and ATP-biotin (Figure 4B and C). The docking studies suggest that APB is a potential kinase cosubstrate But the long distance between the g-phosphate of APB and K168 suggests that APB may be a less efficient cosubstrate compared to ATP or ATP-biotin. 1 2 3 4 5 6 7 HL + -
DHL
NHS biotin
APB
50% TFA
ATP-biotin A
K72
K168
Mg2+ B C
Acknowledgement
We Thank NIH (GM079529),
and
Wayne State University for Funding
Figure 4: Crystal structure image of the catalytic active site of PKA kinase (pdb: 4DH1) docked with APB (A ), ATP-biotin (B), and ATP. Each atom of APB, ATP biotin, and ATP is color-coded (C = green; H grey; N = blue; O = red; p = orange). APB , ATP-biotin, and ATP were docked into the active site of PKA using AutoDock 4.2. Catalytic amino acids (K72 and K168) were shown. The generated data was analyzed by both Auto dock tools and PyMOL (Schrodinger, LLC).