A specific fluorescent probe for protein histidine phosphatase activity

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A specific fluorescent probe for protein histidine phosphatase activity Yigun Choi, Son Hye Shin, Hoyoung Jung, Ohyeon Kwon, Jeong Kon Seo, and Jung-Min Kee ACS Sens., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 26, 2019

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A specific fluorescent probe for protein histidine phosphatase activity Yigun Choi,1,§ Son Hye Shin,1,§ Hoyoung Jung,1 Ohyeon Kwon,1 Jeong Kon Seo,2,* and Jung-Min Kee1,* 1

Department of Chemistry,

2

UNIST Central Research Facilities (UCRF), Ulsan National

Institute of Science and Technology (UNIST), Ulsan 44919, Korea *

Corresponding author: [email protected] (J.-M. Kee) & [email protected] (J. K. Seo)

§

These authors contributed equally.

KEYWORDS Fluorescent

probe,

phosphohistidine

phosphatase,

PHPT1,

chelation-enhanced

fluorescence, enzyme kinetics

ABSTRACT Protein histidine phosphorylation plays a vital role in cell signaling and metabolic processes, and phosphohistidine (pHis) phosphatases such as protein histidine phosphatase 1 (PHPT1) and LHPP have been linked to cancer and diabetes, making them novel drug targets and biomarkers. Unlike the case for other classes of phosphatases, further studies of PHPT1 and other pHis phosphatases have been hampered by the lack of specific activity assays in complex biological mixtures. Previous methods relying on radiolabeling are hazardous and technically laborious, and small molecule phosphatase probes are not selective towards pHis phosphatases. To address these issues, we herein report a fluorescent probe based on chelation-enhanced fluorescence (CHEF) to continuously measure the pHis phosphatase activity of PHPT1. Our probe exhibited excellent sensitivity and specificity towards PHPT1, enabling the first specific measurement of PHPT1 activity in cell lysates. Using this probe, we also obtained more physiologically relevant kinetic parameters of PHPT1, overcoming the limitations of previously used methods.

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Histidine phosphorylation is one of the less studied forms of protein phosphorylation, in contrast to the phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues.1 This is rather striking because histidine phosphorylation was discovered over 50 years ago, much earlier than the now famous Tyr phosphorylation, which was found in 1979.2 Over time, histidine phosphorylation has been implicated in various physiological phenomena, including cell signaling, metabolism, and immune responses. 3 For example, the role of histidine kinases and phosphatases has been well documented in the context of twocomponent signaling systems in bacteria, fungi, and plants.4 However, the mammalian counterparts of these proteins have remained relatively unexplored. 5 To date, the only identified mammalian histidine kinases are isoforms of nuclear diphosphate kinase (NDPK). Moreover, the first mammalian phosphatase PHPT1, dedicated to dephosphorylation of pHis was discovered as recently as in 2002.6 The enzyme PHPT1 dephosphorylates a number of pHis target proteins such as TRPV57 and KCa3.18 ion channels, ATP-citrate lyase (ACL),

9

and subunit of the heterotrimeric G protein,

10

thereby regulating their functions. 11 PHPT1 is also overexpressed in renal cancer, 12 hepatocellular carcinoma13, and lung cancer14 cells, and its knockdown led to reduced cell proliferation or tumorigenesis, making it an attractive potential target for anticancer therapy. Most recently, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), another mammalian pHis phosphatase, was reported as a tumor suppressor in liver cancer.15 It has also been regarded as a key risk factor for depression16 and testicular germ cell tumor.17 However, further studies on pHis-specific phosphatases have been hampered by the lack of suitable research tools. Unlike other types of phosphorylation, it is not trivial to experimentally monitor the degree of histidine phosphorylation in a substrate, due to its instability in acidic solutions and the resultant incompatibility with most biochemical methods to study other types of phosphorylation.18 The 32P-radiolabeling19 method has been traditionally used, but it is hazardous and not specific to pHis. Other methods employing nuclear magnetic resonance (NMR) spectroscopy, 20 mass spectrometry (MS), 21 or malachite-green dye 22 have also been reported, and pHis-specific antibodies have been recently developed to monitor pHis formation and degradation in target proteins or peptides.23 While these methods have circumvented the use of radioisotopes, they are still


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laborious end-point assays and less desirable for kinetic studies and high-throughput screening of potential pHis phosphatase inhibitors. Fluorescence or colorimetry-based probes which allow optical monitoring of the phosphorylation state of the substrate would be perfectly suitable for kinase and phosphatase activity assays.24,25 Such methods would allow continuous real-time measurement of the enzymatic reaction leading to high-throughput screening in a plate format. While pnitrophenyl phosphate (pNPP) and 6,8-difluoromethylumbelliferyl phosphate (DiFMUP), chromogenic or fluorogenic substrates for acid and alkaline phosphatases, have also been employed in pHis phosphatase assays,26 these compounds are inherently non-selective and unable to distinguish PHPT1 activity from other phosphatases. To address these issues, we have developed specific fluorescent chemical probes for real-time monitoring of PHPT1 activity, as reported here. Our pHis-based probes were successfully used to characterize the kinetic parameters of PHPT1, which are more physiologically relevant than previously reported values. Significantly, our probe was highly sensitive and specific towards PHPT1 when tested across a panel of phosphatases, to enable the first specific measurement of PHPT1 activity in cell lysates, which is impossible with previously used methods.

RESULTS

The design of our probe relies on the chelation-enhanced fluorescence (CHEF) effect of the sulfonamidoxine (Sox) fluorophore, pioneered by Imperiali and coworkers. 27 In the absence of phosphorylation, Sox does not show any fluorescence, but upon phosphorylation of an adjacent amino acid residue, it exhibits strong fluorescence due to the intramolecular chelation of Mg2+ between the nascent phosphate and Sox. These CHEF-based probes were initially developed as fluorogenic Ser/Thr or Tyr kinase activity probes but can also be utilized for measuring protein serine/threonine phosphatase (PSP) as well as protein tyrosine phosphatase (PTP) activities.28 Inspired by these reports, we designed our pHis phosphatase activity probes employing Sox (Scheme 1A). Specifically, we attached Sox to the peptide derived from histone H4 sequence, a previously reported peptide substrate used in the kinetic characterization of PHPT1.20 The synthesis was initiated by standard Fmoc-based solid-phase peptide synthesis (SPPS). To selectively install Sox via alkylation onto a cysteine (Cys) residue to form Cys-


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Sox (CSox), a Cys derivative with a hyper acid-labile protecting group (4monomethoxytrityl, Mmt) was used. After the assembly of the desired peptide sequence, the thiol group in the Cys residue was selectively unmasked by mild acid treatment. Subsequent on-resin alkylation with Sox-Br, however, turned out to be less straightforward than expected. While we obtained the desired product using 1,1,3,3-tetramethylguanidine (TMG) as a base, following published protocols,29 we observed incomplete alkylation even after complete consumption of excess Sox-Br during the reaction, suggesting the decomposition of Sox-Br under the reaction conditions. Indeed, the test reaction incubating Sox-Br with TMG resulted in degradation of Sox-Br into multiple unidentified products (data not shown). Accordingly, we changed the base to N,N-diisopropylethylamine (DIPEA), and the on-resin alkylation proceeded to completion, after which the peptide was cleaved from the resin and purified to yield the non-phosphorylated probe, named as Sox-H4 (52% yield) (Scheme 1B).

Scheme 1. (A) Design of the CHEF-based fluorescent probe for pHis phosphatases. (B) Synthesis of Sox-H4P.


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In CHEF-based phosphorylation probes, it is crucial to optimize the position of the CSox moiety for most efficient fluorescence properties since the intramolecular chelation is dependent on the distance and geometry between Sox and the phosphoryl group. Accordingly, we prepared a series of peptide probes by varying the position of Sox around the target His site (Table 1). These peptides were specifically phosphorylated on the histidine residue using potassium phosphoramidate (PPA) and purified by high-performance liquid chromatography (HPLC) to yield the phosphorylated probe, named as Sox-H4P (26% yield). The incorporation and precise location of CSox and pHis in each peptide probe was confirmed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) (Supporting Information). Both 1H- and

31

P-NMR data are consistent with a 3-pHis isomer, which is

thermodynamically favored over 1-pHis (Figure S5). Despite the known instability of pHis, we were able to store Sox-H4P without appreciable dephosphorylation when frozen at pH 9. To our delight, all of our Sox-H4P probes exhibited increased fluorescence compared to the nonphosphorylated Sox-H4 (Table 1). Sox-H4P(-2), the probe with CSox located at the (2) position of pHis, showed the most distinct fluorescence change. In previous studies on the Ser/Thr/Tyr systems, the (-2) position was also found to be optimal.28,30 Sox-H4P(-2) showed lower Kd value (102 ± 9 mM) towards Mg2+ than Sox-H4(-2) (177 ± 30 mM), which implies that phosphorylation of our probe leads to higher affinity for Mg2+ (Figure 1A). The fold change in fluorescence between Sox-H4(-2) and Sox-H4P(-2) was the largest at 20 mM of MgCl2 (Figure 1B). The fluorescence also showed a linear relationship with the degree of phosphorylation and probe concentrations, independently quantified by HPLC peak area, thereby allowing direct optical quantification of phosphorylation and dephosphorylation events (Figure 1C, D). Table 1. Optical properties of Sox-H4P series at different CSox positions. Fold

Peptide substrate sequence Entry

increase in N

-5

-4

-3

-2

-1

0

+1

+2

+3

+4

C

Sox-H4(-2)

Ac

G

G

A

CSox

R

H

R

K

V

L

NH2

3.0 ± 0.1

Sox-H4(-1)

Ac

G

G

A

K

CSox

H

R

K

V

L

NH2

1.6 ± 0.1

Sox-H4(+1)

Ac

G

G

A

K

R

H

CSox

K

V

L

NH2

1.5 ± 0.1

Sox-H4(+2)

Ac

G

G

A

K

R

H

R

CSox

V

L

NH2

2.6 ± 0.2

Sox-H4(+3)

Ac

G

G

A

K

R

H

R

K

CSox

L

NH2

2.0 ± 0.1


fluorescence

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Figure 1. (A) Measurement of Mg2+ dissociation constant for Sox-H4(-2) and Sox-H4P(-2). (B) Fluorescence increase (fold) between Sox-H4(-2) and Sox-H4P(-2) at various [Mg2+]. (C) Linearity between the fluorescence increase and the degree of phosphorylation of Sox-H4(2). (D) Linearity between the fluorescence increase and concentrations of Sox-H4(-2) and Sox-H4P(-2).

With the optimized probe Sox-H4P(-2) in hand, we proceeded to monitor enzymatic activity assays of pHis phosphatases. Gratifyingly, Sox-H4P(-2) demonstrated significant fluorescence decay over time, upon incubation with PHPT1 (Figure 2 and Figure S1). As a negative control, an inactive mutant of PHPT1 (H53A) was also assayed, which showed negligible activity towards our probe. These results show that our probe is indeed dephosphorylated by PHPT1, and it can be used to monitor the phosphatase activity in real time.


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Figure 2. Continuous measurement of pHis dephosphorylation by PHPT1 using Sox-H4P(2).

Reaction kinetic parameters of PHPT1-mediated pHis dephosphorylation were easily measurable using this probe. The progress curves of the dephosphorylation reaction were well fitted with pseudo-first order kinetics for a range of Sox-H4P concentrations (Figure 3A), and the Michaelis-Menten kinetic parameters were calculated from these curves (Figure 3B). Michaelis-Menten parameters obtained using non-pHis small molecule substrates pNPP31 and DiFMUP have been previously reported (Table 2). Our results (KM = 26.89 ± 4.71 µM, kcat = 3.06 ± 0.26 s-1, kcat/KM = (1.14 ± 0.30) × 105 M-1s-1) differ from previously reported values by orders of magnitude, demonstrating the limitation of the use of non-pHis small molecule substrates in the kinetic characterization of protein pHis phosphatases (Table 2). Our assay can be performed with much lower concentrations of enzyme (~10 nM) compared to the small-molecule based assays which require 100-200 nM of PHPT1. Interestingly, the kinetic parameters for PHPT1 are also drastically different from those reported using an analogous histone H4-derived pHis peptide (Table 2).20 However, in these NMR-based kinetic experiments, millimolar concentration (~74 mM) of potassium phosphoramidate (PPA) was also present in the reaction. Since PPA itself is also a substrate for PHPT1, it is likely that PPA competed with the pHis substrate for PHPT1, leading to a significant underestimation of the catalytic efficiency towards pHis. The large apparent KM (1.1 mM) for the pHis substrate supports this hypothesis (Table 2). In contrast, Sox-H4P assays do not contain PPA, and hence the kinetic parameters calculated from these assays are


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more biologically relevant. While the discrepancy between the two studies can be potentially attributed to the substrate sequence difference (AKRpHKV vs. GGA(CSox)RpHRKVL), the NMR study showed similar kinetic parameters for significantly different peptide substrates (AKRpHKV vs. VIFIEpHAKRKG). Therefore, we believe the sequence difference alone cannot explain the ~103-fold differences in the kinetic parameters.

Figure 3. (A) Fluorescence assay of PHPT1 activity at various concentrations of Sox-H4P. (B) Michaelis-Menten saturation curve for PHPT1-catalyzed dephosphorylation of Sox-H4P.

Table 2. Michaelis-Menten kinetic parameters of PHPT1 using different substrates. KM (µM)

kcat (s-1)

kcat / KM (M-1s-1)

Sox-H4P(-2)

26.89 ± 4.71

3.06 ± 0.26

(1.14 ± 0.30) × 105

Histone H4-pHis18 peptidea

1100 ± 450

0.11 ± 0.01

100

Potassium phosphoramidatea

7100 ± 1300

0.37 ± 0.01

52

pNPPb

7400 ± 100

0.35 ± 0.02

47 ± 3

220 ± 30

0.39 ± 0.02

1800 ± 200

DiFMUPc a

Values obtained from reference 20.

b

Values obtained from reference 31.

c

Values

obtained from reference 26.

We also used our assay system to evaluate potential inhibitor candidates of PHPT1. Since there are no known inhibitors for PHPT1, we tested several broad-spectrum phosphatase inhibitors. Interestingly, no significant inhibition of PHPT1 activity was


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observed at millimolar concentrations of these compounds (Figure S2). In case of vanadate, glycerophosphate, and pyrophosphate, the fluorescence assay condition had to be slightly modified to compensate for the presumed competition of Mg2+ chelation to these compounds, with the dephosphorylation of Sox-H4P being independently confirmed by HPLC. To validate the specificity of our probe, we tested the pHis phosphatase activity of other known phosphatases as well. Of particular interest was LHPP enzyme since it is one of the few known mammalian pHis phosphatases. LHPP is known to dephosphorylate Hisphosphorylated protein substrates in vitro, including succinic thiokinase and nucleoside diphosphate kinase.32 Interestingly, LHPP did not dephosphorylate Sox-H4P, which is based on histone H4, implying that LHPP might not have promiscuous pHis phosphatase activity, which is consistent with a recent report15 (Figure 4). Furthermore, protein phosphatase 2C isoform alpha (PP2C), also reported to have pHis phosphatase activity to histone H4,33 did not show activity towards our probe. Lack of activity of other phosphatases including placental alkaline phosphatase (ALPP), protein tyrosine phosphatase 1B (PTP1B), and protein arginine phosphatase (YwlE) towards Sox-H4P, suggests that our probes can distinguish PHPT1 activity not only from other pHis phosphatases but also from other classes of phosphatases. This encouraging result suggests that our probe can specifically detect PHPT1 activities in biological samples such as cell lysates.


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Figure 4. Dephosphorylation of Sox-H4P by various phosphatases. Known substrates for each phosphatase are specified below the bars for each phosphatase.

Encouraged by the specificity of Sox-H4P, we next analyzed the native PHPT1 activity in HeLa cell lysates, using our probe. Since PHPT1 was not inhibited by any of the broadspectrum phosphatase inhibitors (see above), the lysate was treated with a mixture of these inhibitors to observe PHPT1 effect alone, by inhibiting other phosphatases. Negligible difference in phosphatase activity towards our probe was observed between inhibitor-treated and non-treated lysates (Figure 5A). On the other hand, DiFMUP assays showed significantly suppressed phosphatase activity in the inhibitor-treated lysate, indicating that DiFMUP mostly detected phosphatases other than PHPT1 (Figure S3). These results strongly support our probe’s unique specificity towards detecting PHPT1 activity in complex biological samples.


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Figure 5. Detection of PHPT1 activity in HeLa cell lysate. I* refers to the inhibitor mixture of sodium fluoride, imidazole and Phosphatase Inhibitor Cocktail III (Sigma-Aldrich). For the green curve, recombinant PHPT1 (100 nM) was exogenously added to the lysate.

In order to directly validate our probe’s specificity towards PHPT1 in complex biological samples, we performed PHPT1 knockdown in HeLa cells and tested with Sox-H4P(-2). Western blots showed efficient (~96%) siRNA knockdown of PHPT1 (Figure 6A). In our fluorescence assays, HeLa cell lysate with knocked down PHPT1 showed about 50% lower phosphatase activity relative to the native and non-targeting control siRNA lysates (Figure 6B and Figure S4), demonstrating that half of the observed pHis phosphatase activity in the native lysate indeed was from PHPT1 (see Discussion).


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Figure 6. (A) Western blot analysis of siRNA-mediated knockdown of PHPT1 in HeLa cells. Non-targeting (NT) siRNAs were employed as a negative control. (B) Fluorescence assay with the HeLa cell lysates before and after PHPT1 knockdown.

DISCUSSION

Mammalian pHis phosphatase PHPT1 has interesting biological functions related to cancer and diabetes, but its detailed studies have been hampered due to the lack of convenient research tools. We have demonstrated that the CHEF-based pHis probe design is applicable to histidine phosphorylation and dephosphorylation studies and can facilitate its functional studies. Indeed, Sox-H4P allowed convenient assaying and kinetic characterization of the pHis phosphatase activity of PHPT1. Interestingly, the Michaelis-Menten values we obtained were significantly different from previously reported ones. While such kinetic difference between small molecule substrates (pNPP and DiFMUP) and peptide-based substrates are well known for other classes of phosphatases, our data also show difference of several orders of magnitude from those measured using similar histone H4-derived peptide substrates for PHPT1.20 Previous kinetic measurements were carried out at millimolar concentrations of phosphoramidate for continuous histidine phosphorylation of the peptide substrate. However, as the authors have demonstrated, phosphoramidate is also a substrate for PHPT1, which probably competed against the target pHis substrate. Our assay condition is free of


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phosphoramidate, and therefore our kinetic data are likely to be more reliable and relevant. When compared with other classes of protein phosphatases, the catalytic efficiency of PHPT1 lies between the efficiency of human serine/threonine phosphatase 2C-alpha (kcat/KM  104 M-1s-1)34 and protein tyrosine phosphatase 1B (kcat/KM  107 M-1s-1)35 towards their pThr- or pTyr-containing phosphopeptide substrates, respectively. According to our in vitro activity assays, Sox-H4P showed excellent specificity towards PHPT1 over other known histidine phosphatases such as LHPP and PP2C. Moreover, other classes of phosphatases we tested did not recognize Sox-H4P as a substrate. The origin of specificity among pHis phosphatases is unclear at this point, although it may be ascribed to the substrate tolerance of PHPT1 since it efficiently dephosphorylated our probe regardless of the CSox location (Figure S1). In comparison, LHPP had relatively limited scope as pHis substrate,15 and histone H4 has not been listed as a substrate. PP2C is known to dephosphorylate pHis in full-length histone H4, but it might not tolerate CSox. Our probe also successfully measured the activity of nanomolar concentrations of PHPT1, presenting ways to specifically monitor PHPT1 activity in complex biological samples. Indeed, incubation of Sox-H4P with HeLa cell lysates showed robust dephosphorylation even in the presence of broad-spectrum phosphatase inhibitors. While DiFMUP, a general phosphatase substrate recently utilized in PHPT1 activity assays,26 was also dephosphorylated by the HeLa lysate, the dephosphorylation was strongly suppressed by general phosphatase inhibitors, suggesting that the measured activity originated from nonPHPT1 phosphatases. PHPT1 knockdown of HeLa cells with siRNA led to significant reduction of PHPT1 expression, and our fluorescent assay showed that the dephosphorylation of Sox-H4P was reduced to 50%, demonstrating that the phosphatase activity was predominantly from PHPT1. Interestingly, although our siRNA knocked down over 95% of PHPT1, about half of the pHis phosphatase activity persisted. This observation implies the presence of unknown pHis phosphatases recognizing Sox-H4P as a substrate in HeLa cells. It is also possible that PHPT1 activity was regulated in the native lysate in currently unknown ways. Both scenarios show the dearth of our current knowledge about protein histidine phosphorylation, and we believe our probe will be useful in further studies of this important post-translational modification (PTM). While our probes were used for the measurement of enzyme activity of pHis phosphatases, this CHEF-based probe design should also be applicable to protein histidine


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kinases. Histidine kinases are well known for their key signaling roles in bacteria, fungi, and plants. Additionally, recent discoveries about mammalian histidine kinases (NDPK isoforms) also warrants further studies in this direction.

CONCLUSION

Herein we have described a fluorescence-based activity probe for monitoring PHPT1 pHis phosphatase activity. Despite the emerging interest in protein histidine phosphorylation in mammalian systems, the lack of proper research tools has deterred further advancement. Our probe provides the first proof-of-concept fluorescence monitoring tool for pHis level changes. While this study was focused on PHPT1 phosphatase activity, our probe design strategy should also be applicable to histidine kinase probes. Further research in this direction is currently underway in our laboratory and will be reported in due course.

EXPERIMENTAL SECTION

Preparation of of Sox-H4P probes Potassium phosphoramidate (PPA) was synthesized according to the published protocols.36 The Sox-modified peptides were synthesized following the literature,37 with slight modifications (see RESULTS). Obtained Sox-H4 peptides were selectively phosphorylated on histidine using PPA to yield the corresponding Sox-H4P peptides. Briefly, Sox-H4 (1 mM) and PPA (200 mM) were dissolved in 1 mL of pH 8 phosphate buffer, and the mixture was incubated at room temperature for 8 hours. Typically, 80~90% of Sox-H4 was phosphorylated to Sox-H4P in 8 hours. The crude reaction mixture was purified by semipreparative reverse-phase HPLC, and the collected fractions were immediately basified to pH 9 with ammonium hydroxide and evaporated to remove the solvent. Purified Sox-H4P stock solution was quantified with HPLC and stored under -80 oC until further use. The sitespecific incorporation of CSox and pHis were validated by LC-MS/MS analyses.

Preparation of the enzymes


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Coding sequences of PHPT1, LHPP, and YwlE were respectively cloned into a pET21a(+) vector, and the plasmids were competent E. coli BL21(DE3) cells. The expressed recombinant proteins were purified by Ni-NTA affinity chromatography. Protein phosphatase 2C isoform alpha (PP2C), human alkaline phosphatase (ALPP), and protein tyrosine phosphatase 1B (PTP1B) were purchased from Sino Biological.

General procedure for real-time phosphatase activity assay with Sox-H4P The phosphatase reactions were carried out with 10 µM of Sox-H4P, 20 mM of MgCl2, and 10 nM of phosphatases in 200 µL of the standard assay buffer (pH 7.5, 50 mM Tris, 150 mM NaCl, 2 mM DTT, and 1 mM EDTA) in a black 96-well plate. Fluorescence measurements were made by excitation at 360 nm and emission at 500 nm at 25 C on either a SpectraMax i3x Multi-Mode Microplate Readers or a SpectraMax M5e Multi-Mode Microplate Readers (Molecular Devices). All assays were performed in triplicates. The instrumental signal fluctuation was corrected with the fluorescence signals obtained with 4methylumbelliferone. At the end of the measurements, the solution in each well was analyzed with HPLC to verify the dephosphorylation.

Determination of kinetic parameters of PHPT1 Dephosphorylation assays were carried out following the general procedure above except for the concentration of Sox-H4P (1, 2, 5, 10, 15, 20, 30, 50, and 70 µM). The raw fluorescence data (AFU) were converted to the concentration of the Sox-H4P probe (µM) using the relationship from Figure 1C and 1D. The data were plotted and fitted for biphasic pseudo-first-order kinetics, (𝑦 = A1 × exp(−𝑥⁄t1 ) + A2 × exp(−𝑥⁄t 2 ) + y0 ), in which the x-axis is time and the y-axis is Sox-H4P concentration. The biphasic fitting was carried out to extract the kinetic parameters of the PHPT1-catalyzed reaction away from the small and fast signal decay observed in the enzyme-free controls (Figure 2). The initial reaction rates were calculated using the first derivative of the “slower phase” at the initial time point (d𝑦⁄d𝑥 |𝑥=0 = − A2 ⁄t 2 ). The initial rates and the probe concentrations were fitted for the Michaelis-Menten equation to obtain the kinetic parameters (Vmax, KM, and kcat).

PHPT1 siRNAs knockdown in HeLa cells Media for HeLa cells at 50% confluence were exchanged to antibiotic-free media (DMEM supplemented with 10% FBS). siRNAs for PHPT1 and a non-targeting pool were


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purchased from Dharmacon. 45 L of 10 M siRNAs stock prepared in siRNA buffer (pH 7.5, 6 mM HEPES, 60 mM KCl, 0.2 mM MgCl2) and 45 L of Lipofectamine® RNAiMAX Reagent (Invitrogen) were diluted in 1.5 mL of Opti-MEM® reduced serum media. The mixtures were incubated for 20 min at room temperature for siRNAs-lipid complexation, and then added to the cells drop-wise. Cells were incubated for 48 hours at 37 C with 5% CO2, harvested, and lysed in 500 L lysis buffer (pH 8, 50 mM Tris, 150 mM NaCl, 1 mM DTT, 2 mM EGTA, 2 mM PMSF) using a water-bath sonicator.

PHPT1 activity assays in HeLa cell lysates The general PHPT1 assays procedure (see above) was applied with some modifications. 10 µM of Sox-H4P(-2) and 20 mM of MgCl2 were prepared in the standard assay buffer in a 96-well black plate. To see only PHPT1 effect from the lysate by inhibiting other phosphatases, an inhibitor mixture (I*) containing 10 mM of sodium fluoride, 10 mM of imidazole and 1% Phosphatase Inhibitor Cocktail III (Sigma) was treated. 50 µg of HeLa cell lysate (PHPT1-knockdown or control) was added to initiate the reaction, and the fluorescence was monitored. Background signal from the lysate was subtracted from the fluorescence signal obtained with non-phosphorylated Sox-H4(-2) in the lysate. For DiFMUP assays, the protocol in the literature26 was followed with some modifications. 10 µM of DiFMUP and 20 mM of MgCl2 were prepared with optimized buffer (pH 8, 50 mM HEPES, 10 mM NaCl, 0.5 mM DTT) in a 96-well black plate. Fluorescence measurements were made by excitation at 350 nm and emission at 455 nm on a microplate reader.

SUPPORTING INFORMATION

Supplemental figures (Figure S1 to Figure S5), detailed experimental procedures, and characterization data for the probes.

ACKNOWLEDGEMENT


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This research was supported by UNIST (1.140080.01) and National Research Foundation of Korea (2015R1C1A1A02036405). We thank Prof. Hyun-Woo Rhee for helpful discussions and equipment support. We also thank Ji Won Park and Gwangsu Yoon for their help in substrate synthesis.

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e enzymatic activity in unfractionated cell lysates using CSox-based sensors. Curr. Protoc. Chem. Biol. 2014, 6, 135-156.

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A specific fluorescent probe for protein histidine phosphatase activity

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