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Phosphorylation weakens but does not inhibit membrane binding and clustering of K-Ras4B Si-Yu Zhang, Benjamin Sperlich, Fang-Yi Li, Samy Al-Ayoubi, Hong-Xue Chen, YuFen Zhao, Yan-Mei Li, Katrin Weise, Roland Winter, and Yong-Xiang Chen ACS Chem. Biol., Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 29, 2017

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Figure 1. Characterization of synthetic phosphorylated and farnesylated K-Ras4B Protein-OP-SF 4. a) SDSPAGE analysis of K-Ras4B thioester 1 (lane 1), ligation product Protein-OP-SF 4 (lane 2), and Protein-OH-SF 6 as a control (lane 3). b) ESI-MS spectra of K-Ras4B Protein-OP-SF 4 (Mcalcd=21482 Da). The inset shows the original mass spectrum of Protein-OP-SF 4 before deconvolution. c) CD spectra of recombinant K-Ras4B thioester 1 and synthetic K-Ras4B proteins; d) Fluorescence polarization assay of synthetic K-Ras4B ProteinOP-SF 4 loaded with mant-GppNHp upon addition of Raf-RBD with a GST tag. The changes in fluorescence polarization were fitted to Equation 1 (SI) using Grafit (Erithracus software, Horley, UK) to yield the KD value. 57x49mm (300 x 300 DPI)

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Figure 2. a) Overall rotational-correlation times of Bodipy-labeled K-Ras4B proteins. Results are shown for GDP-loaded or GppNHp-loaded Bodipy-Protein-OP-SF 7 and Bodipy-Protein-OH-SF 8 in the absence and presence of anionic lipid vesicles. b) IRRA spectra for K-Ras4B Protein-OH-SF 6 or Protein-OP-SF 4 in interaction with an anionic lipid raft monolayer at high (π ≈ 27 mN m−1, panels ii and iii, respectively) and low (π ≈ 13 mN m−1, panels v and vi, respectively) initial surface pressure. Spectra of the amide-I’ region of K Ras4B were recorded at the respective positions of the surface pressure versus time curve, as indicated by the dips in the surface pressure profiles (i and iv). All spectra were recorded with p-polarized light at an angle of incidence of 35° and at T = 20 ± 0.5 °C; the surface area was held constant. The band at 1735 cm−1 indicates the lipid C=O stretching vibration. c) Fluorescence polarization assays of PDEδ labeled with FITC upon addition of K-Ras4B Protein-OP-SF 4 or Protein-OH-SF 6. Changes in fluorescence polarization were fitted to Equation 1 (SI) using Grafit (Erithracus software, Horley, UK) to yield the similar KD values. 75x41mm (300 x 300 DPI)


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Figure 3. Fluorescence microscopy images showing the distribution of the fluorescent K-Ras4B protein with the nonhydrolyzable phosphoserine mimic Bodipy-Protein-CP-SF 11 and the non-phosphorylable S181A mutant Bodipy-Protein-A-SF 12 in MDCK cells following the intracellular delivery of proteins (left panels). Fluorescence intensity profiles from along the white lines in the left panels are shown in the right panels. 56x48mm (300 x 300 DPI)



Figure 4. Phosphorylation at Ser181 does not change the distribution and clustering of K Ras4B in heterogeneous membranes. AFM images of a heterogeneous model membrane system that consists of DOPC/DOPG/DPPC/DPPG/Chol at a molar ratio of 20:5:45:5:25 and segregates into liquid-ordered (lo) and liquid-disordered (ld) domains at ambient conditions (upper row). Whole scan areas are shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The formation of protein-enriched domains in the ld phase of the heterogeneous membrane system can be observed after addition of Protein-OH-SF 6 (a) and Protein-OP-SF 4 (b and c) (lower row). 48x35mm (300 x 300 DPI)


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Figure 5. Phosphorylation at Ser181 changes the thermotropic properties of K-Ras4B protein-incorporated membranes. DSC thermograms illustrating the effects of K-Ras4B proteins and peptides with different modifications on the thermotropic properties (gel-fluid lipid phase transition temperature Tm) of DMPC/DMPG large unilamellar vesicles. The main phase transition peaks of DMPC/DMPG LUVs including varying concentrations of Peptide-OH-SF 5, Peptide-OP-SF 2, Protein-OH-SF 6 and Protein-OP-SF 4 (2.5, 5, 10 mol%) are presented in panels a, b, c and d, respectively. 36x20mm (300 x 300 DPI)



338x190mm (96 x 96 DPI)


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Phosphorylation weakens but does not inhibit membrane binding and clustering of K-Ras4B Si-Yu Zhang,† Benjamin Sperlich,‡ Fang-Yi Li,† Samy Al-Ayoubi,‡ Hong-Xue Chen,† Yu-Fen Zhao,† Yan-Mei Li,† Katrin Weise,‡ Roland Winter,*,‡ and Yong-Xiang Chen*,† †

Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China ‡

Physical Chemistry I − Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, D-44227 Dortmund, Germany

ABSTRACT: K-Ras4B is one of the most frequently mutated Ras isoforms in cancers. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated Cterminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B’s cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of KRas4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing non-hydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with non-phosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane, but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B’s distribution and activity.

INTRODUCTION

Ras proteins are membrane-bound small GTPases that act as molecular switches by cycling between the active GTPbound state and the inactive GDP-bound state, resulting in the regulation of a variety of cellular processes including cell growth, survival and differentiation.1-3 Mutations in RAS genes, which render Ras proteins constitutively active, are found in 20-30% of human cancers and are among the most powerful drivers of cancers.4 K-Ras4B is one of the most frequently mutated Ras isoforms. However, efforts from both academia and the pharmaceutical industry to develop therapeutics targeting Ras proteins for clinical use have not been successful.4-8 Hence, finding new mechanisms of regulating K-Ras4B’s activity is of importance in tackling Ras-driven cancers. The signaling activity of K-Ras4B depends on its enrichment level on the inner leaflet of the plasma membrane (PM) and its clustering in membrane microdomains, both of which are mediated mainly by K-Ras4B’s unique hypervariable C-terminal region (HVR).9-12 The HVR of K-Ras4B contains a positively-charged Lys-rich motif upstream of a C-terminal farnesylated and methylated cysteine, which is called the “farnesyl–electrostatic

switch”.13 Recent studies have shown that PM enrichment of K-Ras4B also depends on its association with the farnesyl-binding shuttling protein PDEδ that acts as a guanine nucleotide dissociation inhibitor-like solubilizing factor.9,14 Small molecules that bind to PDEδ have been developed to suppress oncogenic K-Ras4B signaling by altering the cellular localization of K-Ras4B.15 In addition to the constitutive cycles that maintain the steady-state PM enrichment of K-Ras4B, extracellularlytriggered conditional phosphorylation at Ser181 of KRas4B, catalyzed by protein kinases (PKC or PKG), has been found to regulate K-Ras4B’s cellular distribution and activity.16,17 Phosphorylated Ser181 is positioned directly within the polybasic stretch of K-Ras4B’s HVR. This modification was reported to lead to the translocation of K-Ras4B from the PM to endomembranes, thereby converting K-Ras4B from a growth-promoting to a growthsuppressing protein.16,18 However, more recent studies have reached the opposite conclusion, reporting that phosphorylation at Ser181 increases the signaling activity of K-Ras4B instead of suppressing it, and does not lead to a change in the localization of the protein to the PM, but rather affects its distribution within this membrane.17,19-21


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The reasons for these discordant observations remain unclear. The Philips’ group observed the dissociation of K-Ras4B from the PM by using a PKC agonist to stimulate conditional phosphorylation in cells16 or by transfecting a construct encoding an S181E mutant of K-Ras4B in which the glutamic acid was used as a constitutively phosphorylated serine mimic..18,22 Groups that have reported opposite findings concerning K-Ras4B localization upon phosphorylation have used an S181D mutant of K-Ras4B or added a PKG agonist.19-21 Differences in the way phosphorylated K-Ras4B or its mimics are generated in vivo may be one reason why opposite conclusions about the effect of phosphorylation at Ser181 on K-Ras4B distribution and activity have been reached. Novel mechanistic insights into these unsolved questions regarding the role of phosphorylation are needed to enable the development of new therapeutic strategies for targeting oncogenic K-Ras4B. We thus constructed an in vitro system containing fully functional modified K-Ras4B and anionic model biomembranes to generate precise molecular level mechanistic information about the membrane partitioning process. We first developed a highly efficient and facile semi-synthetic strategy for producing full-length K-Ras4B proteins containing native farnesyl, phosphate and methyl modifications. Corresponding KRas4B proteins with non-hydrolyzable phosphonate were also synthesized. Next, using this in vitro system, we found that phosphorylation at Ser181 does not fully inhibit the membrane binding of K-Ras4B but weakens its membrane binding ability, in a manner dependent on membrane fluidity. Although phosphorylation does not affect K-Ras4B’s association with its shuttle protein PDEδ, it seems to facilitate the dissociation of K-Ras4B from the plasma membrane, as shown by a decreasing PM distribution of fluorescent K-Ras4B protein with nonhydrolyzable phosphonate. In addition, we demonstrate that phosphorylation does not change the localization of K-Ras4B in the liquid-disordered (ld) lipid subdomains of the membrane and the formation of clusters, but seems to slightly alter the thermotropic properties of K-Ras4Bincorporated lipid bilayers.

RESULTS AND DISCUSSION

Synthesis of phosphorylated, farnesylated and methylated K-Ras4B proteins. Whereas posttranslationally modified proteins with well-defined structures are often not accessible by common expression techniques, protein total synthesis and semi-synthesis become enabling technologies that largely advance the study on those multimodified proteins.23-25 Although farnesylated K-Ras4B has been accessed to by using protein semi-synthesis26 or protein expression in insect cells27, it is still a significant challenge to prepare structure-defined K-Ras4B protein bearing both native phosphate and farnesyl groups on a preparative scale. Herein, we report the first synthesis of K-Ras4B protein embodying phosphate, farnesyl and methyl moieties by a combination of expressed protein ligation (EPL) and lipopeptide synthesis (Scheme 1). The truncated K-Ras4B protein core thioester (K-Ras4B1-174 thioester 1) was generated by intein-mediated thiolysis

following a previously reported method26. Chemical synthesis of the C-terminal peptide (Peptide-OP-SF 2) was difficult due to the acid lability of the farnesyl group and base lability of the protected phosphoserine. Scheme 1. Synthesis of phosphorylated, farnesylated and methylated K-Ras4B proteins

Our previous synthetic strategy for farnesylated and methylated peptide26 is not suitable for Peptide-OP-SF 2, because the farnesyl group incorporated into peptide on resin cannot tolerate the high concentration of TFA commonly used for peptide cleavage, which is particularly often required for the deprotection of phosphoryl ester. We thus developed a facile scheme that allowed the incorporation of multiple modifications into the K-Ras4B peptide (Peptide-OP-SF 2) (Scheme 1). The peptide without the farnesyl group (Peptide-OP-SH 3) was anchored to an acid-sensitive trityl resin through the side-chain thiol group of the cysteine methyl ester as the previously method reported by Distefano group28. After chain elongation ending with an N-terminal cysteine protected as an S-tert-butyl disulfide using an Fmoc strategy, a solution of 95% TFA released Peptide-OP-SH 3 from the resin by simultaneous cleavage of all side-chain protecting groups including those of phosphate. The crude product was subsequently farnesylated with trans,trans-farnesyl bromide in the presence of zinc acetate28,29 (Scheme 1) and then purified by HPLC to generate the pure target peptide Peptide-OP-SF 2, whose identity and purity was confirmed by ESI-MS and NMR spectroscopy (Supporting Information, Materials and Methods and Figure S1). The fully modified C-terminal peptide 2 was then coupled with K-Ras4B1-174 thioester 1 via a native chemical ligation reaction in a high conversion yield (Figure S2). The resultant product was purified by cation exchange chromatography. The resulting phosphorylated and farnesylated full-length K-Ras4B protein (Protein-OP-SF 4) was characterized by SDS-PAGE and ESI-MS (Figure 1 and S2). In general, ca. 5 mg K-Ras4B protein 4 were obtained using 1 L cell suspension expressing the K-Ras4B-intein fusion



protein. Non-phosphorylated K-Ras4B Peptide-OH-SF 5 and Protein-OH-SF 6 were also prepared (Figure S3) following this new strategy instead of our previously reported one due to the higher yield of peptide synthesis (SI, 48% vs 15%)26, which might be caused by avoiding the deprotection of eight Alloc groups of polylysines by Pd(PPh3)4 on resins. We verified the secondary structure of synthetic Protein-OP-SF 4 by circular dichroism (CD) spectroscopy (Figure 1c). The binding affinity of Protein-OP-SF 4 loaded with fluorescent mant-GppNHp showed a KD value of 16 nM (Figure 1d), close to the KD value of recombinant GppNHp-bound K-Ras4B binding to the Ras-binding domain of Raf (Raf-RBD).30 These results not only indicate that the synthetic K-Ras4B Protein-OP-SF 4 is correctly folded and functional but also demonstrate that phosphorylation and farnesylation in the HVR have no obvious effect on the interaction of K-Ras4B protein with the RBD of Raf, one of Ras downstream effectors. In addition, due to the requirement for fluorescencebased biophysical measurements, Bodipy-Protein-OP-SF 7 and Bodipy-Protein-OH-SF 8 were prepared by ligating Bodipy-labeled K-Ras4B1-174 thioester with Peptide-OP-SF 2 and Peptide-OH-SF 5, respectively (Figure S4).

Figure 1. Characterization of synthetic phosphorylated and farnesylated K-Ras4B Protein-OP-SF 4. a) SDS-PAGE analysis of K-Ras4B thioester 1 (lane 1), ligation product Protein-OP-SF 4 (lane 2), and Protein-OH-SF 6 as a control (lane 3). b) ESI-MS spectra of K-Ras4B Protein-OP-SF 4 (Mcalcd=21482 Da). The inset shows the original mass spectrum of Protein-OP-SF 4 before deconvolution. c) CD spectra of recombinant K-Ras4B thioester 1 and synthetic K-Ras4B proteins; d) Fluorescence polarization assay of synthetic K-Ras4B Protein-OP-SF 4 loaded with mantGppNHp upon addition of Raf-RBD with a GST tag. The changes in fluorescence polarization were fitted to Equa-

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tion 1 (SI) using Grafit (Erithracus software, Horley, UK) to yield the KD value. Synthesis of K-Ras4B proteins containing nonhydrolyzable phosphonate. Phosphorylation at serine is a reversible posttranslational modification wherein the removal of phosphate from a protein is catalyzed by phosphatases, leading to difficulties in in vivo studies on protein phosphorylation due to the instability of the P-O bond in cellulo. Non-hydrolyzable phosphonate mimics offer a powerful tool for the accurate study of phosphorylated proteins in vivo, as proteins containing these phosphonate mimics are inert to phosphatases.31,32 To elucidate the influence of phosphorylation at Ser181 on K-Ras4B’s cellular localization by imaging the proteins delivered into cells, the non-hydrolyzable phosphonomethylene alanine (Pma) was incorporated into the Cterminal peptide of K-Ras4B protein instead of phosphoserine following a similar synthetic route to that of Peptide-OP-SF 2. The resultant Peptide-CP-SF 9 was further coupled with K-Ras4B1-174 thioester 1 to generate Protein-CP-SF 10 bearing Pma, which was then characterized by SDS-PAGE and ESI-MS (Figure S5). Fluorescent KRas4B protein with Pma (Bodipy-Protein-CP-SF 11) was generated by ligating Bodipy-labeled K-Ras4B1-174 thioester 1 with Peptide-CP-SF 7. Fluorescent K-Ras4B protein with a S181A (Bodipy-Protein-A-SF 12) mutation that cannot be phosphorylated at site 181 was prepared as a control. Phosphorylation at Ser181 reduces the membrane binding of K-Ras4B in a membrane fluiditydependent manner. The role that phosphorylation at Ser181 plays in regulating K-Ras4B’s cellular distribution remains controversial, as described above. It is thus of significant interest to probe the influence of the phosphate modification on K-Ras4B’s interaction with lipid membranes and the shuttle protein PDEδ in vitro in a precise manner by using quantitative biophysical techniques. We attempted to compare the membrane binding of phosphorylated (Protein-OP-SF 4) and nonphosphorylated K-Ras4B (Protein-OH-SF 6) using frequency-domain fluorescence anisotropy (FDFA) and infrared reflection absorption (IRRA) spectroscopy. The well-established anionic lipid raft membrane system consisting of DOPC/DOPG/DPPC/DPPG/Chol in a molar ratio of 20:5:45:5:25 was used as a model biomembrane system. 31,38 Anionic lipid vesicles and lipid monolayers segregate into liquid-disordered (ld) and liquidordered (lo) domains at ambient conditions and thus mimic the heterogeneity of the plasma membrane.33 They were prepared according to previously reported procedures.34


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Figure 2. a) Overall rotational-correlation times of Bodipy-labeled K-Ras4B proteins. Results are shown for GDP-loaded or GppNHp-loaded Bodipy-Protein-OP-SF 7 and Bodipy-Protein-OH-SF 8 in the absence and presence of anionic lipid vesicles. b) IRRA spectra for K-Ras4B Protein-OH-SF 6 or Protein-OP-SF 4 in interaction with an anionic lipid raft monolayer at high −1 −1 (π ≈ 27 mN m , panels ii and iii, respectively) and low (π ≈ 13 mN m , panels v and vi, respectively) initial surface pressure. Spectra of the amide-I’ region of K-Ras4B were recorded at the respective positions of the surface pressure versus time curve, as indicated by the dips in the surface pressure profiles (i and iv). All spectra were recorded with p-polarized light at an 1 angle of incidence of 35° and at T = 20 ± 0.5 °C; the surface area was held constant. The band at 1735 cm− indicates the lipid C=O stretching vibration. c) Fluorescence polarization assays of PDEδ labeled with FITC upon addition of K-Ras4B ProteinOP-SF 4 or Protein-OH-SF 6. Changes in fluorescence polarization were fitted to Equation 1 (SI) using Grafit (Erithracus software, Horley, UK) to yield the similar KD values.

We first measured the dynamic properties of K-Ras4B and its membrane interaction using frequency-domain fluorescence anisotropy. The overall rotational correlation time of the Bodipy-labeled K-Ras4B protein, θBodipy-K-Ras4B, is proportional to the size of the fluorophore-labeled protein.34 In comparison with non-phosphorylated KRas4B (θ = 23.99 ± 3 ns), phosphorylated K-Ras4B proteins display slightly smaller θBodipy-K-Ras4B values (θ = 20.90 ± 2, p-value 0.19, GDP-loaded; θ = 20.50 ± 2 ns, p-value 0.17, GppNHp-loaded; without significant difference) in the presence of anionic model raft membranes. Moreover, our data clearly show a significant increase in the rotational correlation times for both phosphorylated and nonphosphorylated K-Ras4B proteins upon addition of large unilamellar vesicles (LUVs) of the anionic model biomembrane, indicating that phosphorylation does not inhibit membrane binding of K-Ras4B (Figure 2a). That the change in θ is indeed due to the binding of K-Ras4B to LUVs was proven in a previous study by performing the same experiment with non-lipidated K-Ras4B, when a rotational correlation time of 13 ns was reported, corresponding to no membrane binding 34,35. The slightly reduced retardation of the rotational dynamics of the membrane-bound phosphorylated K-Ras4B might be explained by diminished electrostatic interactions and thus weaker membrane binding upon phosphorylation, as phosphorylation at Ser181 decreases the positive net charge of the polybasic motif of K-Ras4B.

IRRA spectroscopy experiments provide support for these findings. By simultaneously recording IRRA spectra and surface pressure/time (π/t) isotherms, we were able to study the interaction of K-Ras4B proteins with lipid monolayers composed of the anionic raft mixture (Figure 2b). Upon injection of the proteins underneath the lipid monolayer at an initial surface pressure of ∼27 mN m−1, which reflects the physiological lipid density generally found in lipid membranes,36 an increase in surface pressure of about 5 mN m−1 was detected for nonphosphorylated K-Ras4B. This increase results from insertion of the lipid anchors of farnesylated Ras proteins into the lipid monolayer, as non-lipidated Ras constructs are unable to insert into lipid monolayers and thus exhibit no increase in surface pressure.36 The corresponding amide-I' band is characteristic for accumulated K-Ras4B proteins at the lipid interface (Figure 2b, panel ii). When phosphorylated K-Ras4B was injected, no increase in surface pressure and no amide-I' band could be detected, regardless of the nucleotides bound to Ras. However, at a lower initial surface pressure (π ≈ 13 mN m−1) all proteins were able to bind to the lipid monolayer, as indicated by an increase in π of about 10 mN m−1 and a corresponding amide-I' band at ∼1639 cm−1. Consequently, phosphorylation does not inhibit K-Ras4B membrane binding but makes it dependent on the fluidity of the membrane, requiring a more fluid environment, i.e., a higher confor-



mational lipid disorder, for efficient membrane partitioning. Phosphorylation at Ser181 does not affect the binding affinity between K-Ras4B and PDEδ. PM enrichment of K-Ras4B has been attributed not only to lipid insertion and electrostatic interaction between the “farnesyl–electrostatic switch” and the PM but also to farnesylbinding shuttle proteins like PDEδ.9,14 PDEδ possesses both a hydrophobic pocket that associates with K-Ras4B’s farnesyl moiety and a negatively charged surface that is estimated to interact with K-Ras4B’s poly-Lys motif.14,26 The phosphate at Ser181 might therefore affect the interaction between K-Ras4B and PDEδ by changing the net charge of the switch. In order to address this issue, the binding affinities between FITC-labeled PDEδ and Protein-OP-SF 4 or Protein-OH-SF 6 were determined using a fluorescence polarization assay. As shown in Figures 2c and S6, similar binding affinities indicated that phosphorylation at Ser181 has no significant influence on the interaction between K-Ras4B and PDEδ, regardless of the nucleotides bound to Ras. It may also be deduced that the side chain of Ser181 is most likely not involved in the association of K-Ras4B with PDEδ. Taken together, using in vitro protein-membrane systems, we have demonstrated that phosphorylation at Ser181 does not fully inhibit membrane binding of KRas4B, a decrease of binding affinity is observed upon phosphorylation, only. In addition, we have shown that membrane binding of phosphorylated K-Ras4B is dependent on membrane fluidity, and phosphorylated KRas4B maintains tight association with PDEδ. As a result, phosphorylation may perturb the constitutive spatial cycles of K-Ras4B to some extent by facilitating the dissociation of K-Ras4B from the plasma membrane due to reduced electrostatic interactions between the polybasic stretch with K-Ras4B and the negatively charged plasma membrane. Phosphorylated K-Ras4B protein displays a decreasing PM distribution. In order to further investigate the regulation of phosphorylation at Ser181 on K-Ras4B’s cellular distribution, we planned to image the cells after respectively delivering fluorescent K-Ras4B protein with the non-hydrolyzable phosphoserine mimic BodipyProtein-CP-SF 11, and non-phosphorylable S181A mutant Bodipy-Protein-A-SF 12 into cells. Since the delivery of exogenous peptides and proteins into cells is difficult, versatile methodologies have been developed to achieve this goal, including utilizing cell-penetrating cationic peptides such as polyarginines or polylysines.37 As KRas4B contains a polylysine stretch in the C-terminus, we used pyrenebutyrate as a counteranion to achieve delivery of the proteins directly into the cells. Pyrenebutyrate is a negatively charged and hydrophobic compound, which can interact electrostatically with positively charged peptides and assist positively charged peptide translocation through cell membranes.38,39 After incubation with pyrenebutyrate for 5 minutes, MDCK cells were treated with Bodipy-Protein-CP-SF 11 or Bodipy-Protein-A-SF 12 for another 5 minutes. Afterwards,

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cells were washed with PBS, incubated in fresh medium at 37°C (5% CO2) for 1 h, and then monitored by confocal microscopy. The distribution of Bodipy-Protein-CP-SF 11 and Bodipy-Protein-A-SF 12 in MDCK cells is shown in Figure 3. From the fluorescence intensity profile along the white line in the left panel of Figure 3a, a reduction in PM localization can be observed in Bodipy-Protein-CP-SF 11 treated cells. Thus, we conclude that phosphorylation at Ser181 can facilitate the release of K-Ras4B protein from the plasma membrane.

Figure 3. Fluorescence microscopy images showing the distribution of the fluorescent K-Ras4B protein with the nonhydrolyzable phosphoserine mimic Bodipy-ProteinCP-SF 11 and the non-phosphorylable S181A mutant Bodipy-Protein-A-SF 12 in MDCK cells following the intracellular delivery of proteins (left panels). Fluorescence intensity profiles from along the white lines in the left panels are shown in the right panels. Phosphorylation has no influence on the lateral membrane organization of K-Ras4B. Correct localization to negatively charged plasma membranes and formation of protein clusters in membrane subdomains are essential for K-Ras4B to achieve its signaling function. Our previous results show that unphosphorylated farnesylated K-Ras4B localizes preferentially in liquiddisordered lipid domains of heterogeneous model biomembrane systems, where it forms protein-enriched domains.40 To investigate the influence of phosphorylation on K-Ras4B’s localization and clustering in membrane subdomains, we analyzed the distribution of phosphorylated K-Ras4B Protein-OP-SF 4 in the same heterogeneous anionic model biomembrane system as described above. Tapping-mode atomic force microscopy (AFM) measurements allow imaging of the same sample area before and after the addition of a protein to a solid-supported lipid membrane. Phase separation into lo and ld domains could be observed when the anionic lipid raft membrane was imaged before injecting K-Ras4B protein solution into the AFM fluid cell (Figure 4, upper panels). After incubation of the lipid bilayer with GDP- or GppNHp loaded K-Ras4B Protein-OP-SF 4, the AFM images show the same membrane partitioning behavior of proteins


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independent of nucleotides (Figure 4b and c), i. e., the KRas4B-enriched domains are exclusively localized in the liquid-disordered phase of the membrane. Comparison with non-phosphorylated K-Ras4B Protein-OH-SF 6 as a control (Figure 4a) indicated that phosphorylation does not significantly change the distribution and clustering of K-Ras4B in heterogeneous membranes. However, due to slightly different local protein concentrations in the AFM fluid cell and different lipid domain sizes, the height and lateral dimensions of the protein-enriched domains vary slightly. This is an important finding since a previous study hypothesized that phosphorylation of Ser181 inhibits the formation of K-Ras4B nanoclusters in the plasma membrane.41 While we did not detect significant differences in protein cluster formation upon phosphorylation of K-Ras4B in vitro, Barceló et al. showed that oncogenic K-Ras4B segregates into spatially distinct nanoclusters at the plasma membrane, depending on its phosphorylation status.20 In addition, our results for the membrane binding behavior of phosphorylated K-Ras4B are in agreement with reports that the majority of pseudo-phosphorylated K-Ras4B is still located at the plasma membrane41,42.

Figure 4. Phosphorylation at Ser181 does not change the distribution and clustering of K-Ras4B in heterogeneous membranes. AFM images of a heterogeneous model membrane system that consists of DOPC/DOPG/DPPC/DPPG/Chol at a molar ratio of 20:5:45:5:25 and segregates into liquid-ordered (lo) and liquid-disordered (ld) domains at ambient conditions (upper row). Whole scan areas are shown with a vertical color scale from dark brown to white corresponding to an overall height of 8 nm. The formation of protein-enriched domains in the ld phase of the heterogeneous membrane system can be observed after addition of Protein-OH-SF 6 (a) and Protein-OP-SF 4 (b and c) (lower row). Phosphorylation at Ser181 changes the thermotropic properties of K-Ras4B-incorporated membranes. To yield additional insight into the effects of phosphorylation on K-Ras4B’s interaction with membranes, we used differential scanning calorimetry (DSC) to probe the thermotropic properties of K-Ras4Bincorporated membranes. DSC is a very useful technique for measuring thermally induced phase transitions of membranes, as membranes are highly sensitive to exogenously added compounds.43-46 Thus, monitoring changes in phase transition peaks shown in DSC thermograms can

provide information about the membrane binding ability of proteins. DMPC/DMPG (3:1, molar ratio) anionic large unilamellar vesicles (LUVs) with a main gel-fluid lipid phase transition temperature (Tm) of around 25°C were chosen as a model membrane system, and DSC measurements were recorded for K-Ras4B proteins and their Cterminal peptides with different modification patterns.

Figure 5. Phosphorylation at Ser181 changes the thermotropic properties of K-Ras4B protein-incorporated membranes. DSC thermograms illustrating the effects of KRas4B proteins and peptides with different modifications on the thermotropic properties (gel-fluid lipid phase transition temperature Tm) of DMPC/DMPG large unilamellar vesicles. The main phase transition peaks of DMPC/DMPG LUVs including varying concentrations of Peptide-OH-SF 5, Peptide-OP-SF 2, Protein-OH-SF 6 and Protein-OP-SF 4 (2.5, 5, 10 mol%) are presented in panels a, b, c and d, respectively. First, K-Ras4B peptides of varying concentrations (0, 2.5, 5, 10 mol%) were incubated with DMPC/DMPG LUVs. Peptide-OH-SF 5 and Peptide-OP-SF 2 both markedly reduced the Tm of the membrane system (Figure 5a and 5b). In contrast, peptides lacking a farnesyl moiety did not change the Tm of the membrane (data not shown). This observation is consistent with previous reports stating that the partitioning of farnesylated peptides into the membrane can markedly reduce the Tm of the membrane as it affects the lipid acyl chain packing of the membrane by the unsaturated farnesyl group.44 In addition, we performed DSC scans for DMPC/DMPG LUVs containing increasing concentrations (0, 2.5, 5, 10 mol%) of fulllength K-Ras4B proteins (Protein-OH-SF 6 and ProteinOP-SF 4). In comparison with the DSC thermograms for the peptides, full-length K-Ras4B proteins reduced Tm to different degrees, indicating that the N-terminal part of K-Ras4B affects the partitioning process, probably owing to differences in lateral organization within the membrane plane and the clustering propensity of the protein (Figure 5c and 5d). Moreover, the reduction in Tm caused by Protein-OP-SF 4 was less than that caused by ProteinOH-SF 6 at the same protein concentration, demonstrating that phosphorylation at Ser181 modulates the partitioning process and lateral organization to some extent. In contrast, decreases in Tm induced by adding the same amount of Peptide-OH-SF 5 or Peptide-OP-SF 2 were similar, implying that phosphorylation affects the thermotropic properties of K-Ras4B protein incorporated



membranes due to the involvement of the N-terminal part of protein. Membrane viscosity (fluidity) is a key parameter that characterizes the mechanical properties of membrane and is associated with the diffusion of membrane-binding proteins.47 In order to explore the influence of phosphorylation at Ser181 on the viscosity of K-Ras4B incorporated membranes, the fluorescent probe DPH (1,6diphenylhexatriene) was incorporated into membranes for steady fluorescence emission polarization measurements in the absence and presence of Protein-OH-SF 6 and Protein-OP-SF 4. The resulting polarization value finally yielded the membrane viscosity parameter η according to Equation 2 (SI).48 The two artificial membrane systems used above (DMPC/DMPG vesicles, and DOPC/DOPG/DPPC/DPPG/Chol vesicles) and Hela cell membranes were employed for these measurements. Results shown in Figure S7 show that the incorporation of Protein-OP-SF 4 into the DMPC/DMPG vesicles, DOPC/DOPG/DPPC/ DPPG/Chol vesicles and Hela cell membranes resulted in a higher membrane viscosity compared with Protein-OH-SF 6, probably due to differences in membrane partitioning and dynamics after the respective incorporation of these two proteins.

CONCLUSIONS

In summary, we have successfully developed a highly efficient and facile semi-synthetic strategy for preparing fully functional phosphorylated, farnesylated and methylated K-Ras4B proteins. These proteins are very useful tools for exploring the precise effects of phosphorylation at Ser181 on the cellular distribution and activity of KRas4B. By utilizing these proteins in an in vitro investigation, we found that phosphorylation does not fully inhibit the membrane association of K-Ras4B, but rather weakens its interaction with membranes, in a membrane fluidity-dependent manner. On the other hand, phosphorylation does not affect K-Ras4B’s binding with its cytosolic shuttle protein PDEδ. These results provide a possible explanation for the decreasing PM distribution of phosphorylated K-Ras4B observed in cellulo. Moreover, phosphorylation does not significantly change the localization of K-Ras4B in the fluid-like lipid subdomains of membranes and their propensity to form clusters, but slightly alters the thermotropic properties of K-Ras4Bincorporated lipid bilayers, reflecting their different membrane partitioning propensities. Moreover, the dynamics changes slightly as well as inferred from the timeresolved fluorescence anisotropy data. Those may facilitate modulation of K-Ras4B’s constitutive spatial cycle.

ASSOCIATED CONTENT

Supporting Information This Supporting Information is available free of charge on the ACS Publications website at XXX. Materials and methods, compounds characterization data, including Figure S1-S7 (PDF).

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AUTHOR INFORMATION Corresponding Author * [email protected] [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was supported by grants from the Major State Basic Research Development Program of China (2013CB910700), the National Natural Science Foundation of China (21372140, 21672125) and the Deutsche Forschungsgemeinschaft (DFG, SFB 642). We thank YY Chen from the Institute of Biophysics, Chinese Academy of Sciences for assistance with operating DSC facilities.

REFERENCES (1) Wittinghofer, A., and Waldmann, H. (2000) Ras - A molecular switch involved in tumor formation, Angew. Chem. Int. Ed. 39, 4193-4214. (2) Pylayeva-Gupta, Y., Grabocka, E., and Bar-Sagi, D. (2011) RAS oncogenes: weaving a tumorigenic web, Nat. Rev. Cancer 11, 761774. (3) Milroy L.-G. and Ottmann C. (2014) The Renaissance of Ras, ACS Chem. Biol. 9, 2447-2458. (4) Prior, I. A., Lewis, P. D., and Mattos, C. (2012) A Comprehensive Survey of Ras Mutations in Cancer, Cancer Res. 72, 24572467. (5) Ledford, H. (2015) The Ras Renaissance, Nature 520, 278-280. (6) Spiegel, J., Cromm, P. M., Zimmermann, G., Grossmann, T. N., and Waldmann, H. (2014) Small-molecule modulation of Ras signaling, Nat. Chem. Biol. 10, 613-622. (7) Cox, A. D., Fesik, S. W., Kimmelman, A. C., Luo, J., and Der, C. J. (2014) Drugging the undruggable RAS: Mission Possible?, Nat. Rev. Drug Discov. 13, 828-851. (8) Ostrem, J. M. L., and Shokat, K. M. (2016) Direct smallmolecule inhibitors of KRAS: from structural insights to mechanism-based design, Nat. Rev. Drug Discov. 15, 771-785. (9) Schmick, M., Vartak, N., Papke, B., Kovacevic, M., Truxius, D. C., Rossmannek, L., and Bastiaens, P. I. (2014) KRas localizes to the plasma membrane by spatial cycles of solubilization, trapping and vesicular transport, Cell 157, 459-471. (10) Hancock, J. F. (2003) Ras proteins: different signals from different locations, Nat. Rev. Mol. cell Biol. 4, 373-384. (11) Plowman, S. J., Muncke, C., Parton, R. G., and Hancock, J. F. (2005) H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton, Proc. Natl. Acad. Sci. U.S.A. 102, 15500-15505. (12) Nan, X., Tamgueney, T. M., Collisson, E. A., Lin, L.-J., Pitt, C., Galeas, J., Lewis, S., Gray, J. W., McCormick, F., and Chu, S. (2015) Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway, Proc. Natl. Acad. Sci. U.S.A. 112, 7996-8001.


Page 15 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(13) Ahearn, I. M., Haigis, K., Bar-Sagi, D., and Philips, M. R. (2012) Regulating the regulator: post-translational modification of RAS, Nat. Rev. Mol. Cell Biol. 13, 39-51. (14) Ismail, S. A., Chen, Y. X., Rusinova, A., Chandra, A., Bierbaum, M., Gremer, L., Triola, G., Waldmann, H., Bastiaens, P. I., and Wittinghofer, A. (2011) Arl2-GTP and Arl3-GTP regulate a GDI-like transport system for farnesylated cargo, Nat. Chem. Biol. 7, 942-949. (15) Zimmermann, G., Papke, B., Ismail, S., Vartak, N., Chandra, A., Hoffmann, M., Hahn, S. A., Triola, G., Wittinghofer, A., Bastiaens, P. I., and Waldmann, H. (2013) Small molecule inhibition of the KRAS-PDEdelta interaction impairs oncogenic KRAS signalling, Nature 497, 638-642. (16) Bivona, T. G., Quatela, S. E., Bodemann, B. O., Ahearn, I. M., Soskis, M. J., Mor, A., Miura, J., Wiener, H. H., Wright, L., Saba, S. G., Yim, D., Fein, A., Perez de Castro, I., Li, C., Thompson, C. B., Cox, A. D., and Philips, M. R. (2006) PKC regulates a farnesylelectrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis, Mol. Cell 21, 481493.

(26) Chen, Y. X., Koch, S., Uhlenbrock, K., Weise, K., Das, D., Gremer, L., Brunsveld, L., Wittinghofer, A., Winter, R., Triola, G., and Waldmann, H. (2010) Synthesis of the Rheb and K-Ras4B GTPases, Angew. Chem. Int. Ed. 49, 6090-6095. (27) Gillette, W. K., Esposito, D., Blanco, M.A., Alexander, P., Bindu, L., Bittner, C., Chertov, O., Frank, P.H., Grose, C., Jones, J.E., Meng, Z., Perkins, S., Van, Q., Ghirlando, R., Fivash, M., Nissley, D.V., McCormick, F., Holderfield, M.A., Stephen, G. (2015) Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions, Sci. Rep. 5, 15916. (28) Diaz-Rodriguez, V., Mullen, D. G., Ganusova, E., Becker, J. M., and Distefano, M. D. (2012) Synthesis of peptides containing C-terminal methyl esters using trityl side-chain anchoring: application to the synthesis of a-factor and a-factor analogs, Organic Lett. 14, 5648-5651. (29) Xue, C. B., Caldwell, G. A., Becker, J. M., and Naider, F. (1989) Total Synthesis of the Lipopeptide a-Mating Factor of Saccharomyces Cerevisiae, Biochem. Biophys. Res. Commun. 162, 253-257.

(17) Cho, K.-j., Casteel, D. E., Prakash, P., Tan, L., van der Hoeven, D., Salim, A. A., Kim, C., Capon, R. J., Lacey, E., Cunha, S. R., Gorfe, A. A., and Hancock, J. F. (2016) AMPK and eNOS signaling regulates K-Ras plasma membrane interactions via cGMP-dependent Protein Kinase 2, Mol. Cell Biol. 36, 3086-3099

(30) Herrmann, C., Horn, G., Spaargaren, M., and Wittinghofer, A. (1996) Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor, J. Biol. Chem. 271, 6794-6800.

(18) Sung, P. J., Tsai, F. D., Vais, H., Court, H., Yang, J., Fehrenbacher, N., Foskett, J. K., and Philips, M. R. (2013) Phosphorylated K-Ras limits cell survival by blocking Bcl-xL sensitization of inositol trisphosphate receptors, Proc. Natl. Acad. Sci. U.S.A. 110, 20593-20598.

(31) Szewczuk, L. M., Tarrant, M. K., and Cole, P. A. (2009) Chapter 1 Protein Phosphorylation by Semisynthesis, Methods Enzymol. 462, 1-24.

(19) Alvarez-Moya, B., Lopez-Alcala, C., Drosten, M., Bachs, O., and Agell, N. (2010) K-Ras4B phosphorylation at Ser181 is inhibited by calmodulin and modulates K-Ras activity and function, Oncogene 29, 5911-5922. (20) Barcelo, C., Paco, N., Beckett, A. J., Alvarez-Moya, B., Garrido, E., Gelabert, M., Tebar, F., Jaumot, M., Prior, I., and Agell, N. (2013) Oncogenic K-ras segregates at spatially distinct plasma membrane signaling platforms according to its phosphorylation status, J. Cell Sci. 126, 4553-4559.

(32) Klingberg, R., Jost, J. O., Schumann, M., Gelato, K. A., Fischle, W., Krause, E., and Schwarzer, D. (2015) Analysis of phosphorylation-dependent protein-protein interactions of histone h3, ACS Chem. Biol. 10, 138-145. (33) Kapoor, S., Werkmueller, A., Denter, C., Zhai, Y., Markgraf, J., Weise, K., Opitz, N., and Winter, R. (2011) Temperaturepressure phase diagram of a heterogeneous anionic model biomembrane system: Results from a combined calorimetry, spectroscopy and microscopy study, Biochim. Biophys. Acta.Biomembrane 1808, 1187-1195.

(21) Barcelo, C., Paco, N., Morell, M., Alvarez-Moya, B., BotaRabassedas, N., Jaumot, M., Vilardell, F., Capella, G., and Agell, N. (2014) Phosphorylation at Ser-181 of oncogenic KRAS is required for tumor growth, Cancer Res. 74, 1190-1199.

(34) Weise, K., Kapoor, S., Werkmuller, A., Mobitz, S., Zimmermann, G., Triola, G., Waldmann, H., and Winter, R. (2012) Dissociation of the K-Ras4B/PDEdelta complex upon contact with lipid membranes: membrane delivery instead of extraction, J. Am. Chem. Soc. 134, 11503-11510.

(22) Quatela, S. E., Sung, P. J., Ahearn, I. M., Bivona, T. G., and Philips, M. R. (2008) Analysis of K‐Ras Phosphorylation, Translocation, and Induction of Apoptosis, Methods in Enzymol. 439, 87-102.

(35) Werkmuller, A., Triola, G., Waldmann, H., and Winter, R. (2013) Rotational and translational dynamics of ras proteins upon binding to model membrane systems, Chemphyschem 14, 3698-3705.

(23) Dawson, P. E., Muir, T. W., Clark-Lewis, I., and Kent, S. B. (1994) Synthesis of proteins by native chemical ligation, Science 266, 776-779.

(36) Kapoor, S., Weise, K., Erlkamp, M., Triola, G., Waldmann, H., and Winter, R. (2012) The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction, Eur. Biophys. J. Biophys. Lett. 41, 801-813.

(24) Flavell, R. R., and Muir, T. W. (2009) Expressed protein ligation (EPL) in the study of signal transduction, ion conduction, and chromatin biology, Acc. Chem. Res. 42, 107-116. (25) Muller, M. M., and Muir, T. W. (2015) Histones: at the crossroads of peptide and protein chemistry, Chem. Rev. 115, 22962349.

(37) David, Y., Vila-Perello, M., Verma, S., and Muir, T. W. (2015) Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins, Nat. Chem. 7, 394-402. (38) Perret, F., Nishihara, M., Takeuchi, T., Futaki, S., Lazar, A. N., Coleman, A. W., Sakai, N., and Matile, S. (2005) Anionic



Page 16 of 17

fullerenes, calixarenes, coronenes, and pyrenes as activators of oligo/polyarginines in model membranes and live cells, J. Am. Chem. Soc. 127, 1114-1115.

(43) Rowat, A. C., Keller, D., and Ipsen, J. H. (2005) Effects of farnesol on the physical properties of DMPC membranes, Biochim. Biophys. Acta. 1713, 29-39.

(39) Takeuchi, T., Kosuge, M., Tadokoro, A., Sugiura, Y., Nishi, M., Kawata, M., Sakai, N., Matile, S., and Futaki, S. (2006) Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate, ACS Chem. Biol. 1, 299-303.

(44) Rowat, A. C., Brask, J., Sparrman, T., Jensen, K. J., Lindblom, G., and Ipsen, J. H. (2004) Farnesylated peptides in model membranes: a biophysical investigation, Eur. Biophys. J. 33, 300-309.

(40) Weise, K., Kapoor, S., Denter, C., Nikolaus, J., Opitz, N., Koch, S., Triola, G., Herrmann, A., Waldmann, H., and Winter, R. (2011) Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms, J. Am. Chem. Soc. 133, 880-887. (41) Plowman, S. J., Ariotti, N., Goodall, A., Parton, R. G., and Hancock, J. F. (2008) Electrostatic interactions positively regulate K-Ras nanocluster formation and function, Mol. Cell Biol. 28, 4377-4385. (42) Lopez-Alcala, C., Alvarez-Moya, B., Villalonga, P., Calvo, M., Bachs, O., and Agell, N. (2008) Identification of essential interacting elements in K-Ras/calmodulin binding and its role in KRas localization, J. Biol. Chem. 283, 10621-10631.

(45) Joanne, P., Galanth, C., Goasdoue, N., Nicolas, P., Sagan, S., Lavielle, S., Chassaing, G., El Amri, C., and Alves, I. D. (2009) Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry, Biochim. Biophys. Acta. 1788, 1772-1781. (46) Canadas, O., and Casals, C. (2013) Differential scanning calorimetry of protein-lipid interactions, Methods Mol. Biol. 974, 55-71. (47) Goodwin, J. S., Drake, K. R., Remmert, C. L., and Kenworthy, A. K. (2005) Ras diffusion is sensitive to plasma membrane viscosity, Biophys. J. 89, 1398-1410. (48) Yuan, H.-L., Liu, X.-L., and Liu, Y.-J. (2014) Solanum Nigrum Polysaccharide (SNL) Extract Effects in Transplanted Tumorbearing Mice - Erythrocyte Membrane Fluidity and Blocking of Functions, Asian Pac. J. Cancer Prev. 15, 10469-10473.


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