Real time characterization of the MAPK pathway using native mass spectrometry

Abstract

The MAPK pathway is a crucial cell-signaling cascade that is composed of RAS, MEK, BRAF, and ERK, which serves to connect extracellular signals to intracellular responses. Over-activating mutations in the MAPK pathway can lead to uncontrolled cell growth ultimately resulting in various types of cancer. While this pathway has been heavily studied using a battery of techniques, herein we employ native mass spectrometry (MS) to characterize the MAPK pathway, including nucleotide, drug, and protein interactions. We utilize native MS to provide detailed insights into nucleotide and drug binding to BRAF complexes, such as modulation of nucleotide binding in the presence of MEK1. We then demonstrate that different CRAF segments vary in their complex formation with KRAS, with the addition of the cysteine rich domain (CRD) enhancing complex formation compared to Ras binding domain (RBD) alone. We report differences in KRAS GTPase activity in the presence of different RAF segments, with KRAS exhibiting significantly enhanced nucleotide turnover when bound to CRAF fragments. We use ERK2 as a downstream readout to monitor the MAPK phosphorylation cascade. This study demonstrates the utility of native MS to provide detailed characterization of individual MAPK pathway components and monitor the phosphorylation cascade in real time.

Fig. 1. Biophysical characterization of BRAF-MEK complex.

A Mass spectrum of (BRAF)(14-3-3)₂ in maroon and (MEK1)(BRAF)(14-3-3)₂ in dark blue at 2 µM. Deconvoluted mass spectrum shown in inset. B Isolation of 29⁺ ion (7693 m/z) and application of collisional activation energy (260 eV) revealed two components of the 14-3-3 dimer, MEK1 alone, and both complexes of (MEK1)(BRAF)(14-3-3)₁ varying in the 14-3-3 bound. C Deconvolution of mass spectra shown in (B). D Addition of 5 µM of MEK1 shifts population towards the (MEK1)(BRAF)(14-3-3)₂ complex. E The addition of 25 µM ATP (green) to the sample from panel A displayed a 503 ± 2 Da shift to the (BRAF)(14-3-3)₂ complex. The addition of 25 µM ATP to the sample from panel A displayed one and two 530 ± 7 Da shifts to the (MEK1)(BRAF)(14-3-3)₂ complex. F Addition of 25 µM ATP and 5 µM of GDC-0623 to the sample from panel A displayed drug binding (yellow) of 475 Da to (MEK1)(BRAF)(14-3-3)₂ complex.

Fig. 2. Impacts of KRAS bindings based on different segments of RAF.

A Mass spectrum of CRAF-RBD (light orange) and KRAS•GTP (light green) mixed at 2 µM to 0.5 µM ratio, respectively, to form a complex with CRAF-RBD and KRAS•GTP. B Percentage of KRAS, Q61H (dark purple), G12C (dark green), and G13D (light pink) bound to CRAF-RBD. C Mass spectrum of 2 µM CRAF-RBDCRD (dark orange) mixed with 0.5 µM KRAS•GTP. D Percentage of KRAS, Q61H, G12C, and G13D bound to CRAF-RBDCRD. E Mass spectrum of (BRAF)(14-3-3)₂ and (MEK1)(BRAF)(14-3-3)₂ at 2 µM in the presence of 2 µM KRAS. F Deconvoluted mass spectra shown in (E). G Percentage of KRAS, Q61H, G12C, and G13D bound individually to (BRAF)(14-3-3)₂ and (MEK1)(BRAF)(14-3-3)₂. Two-tailed t-test was performed, with one asterisk denoting p values < 0.05 and two asterisks denoting p-values < 0.01. The error bar defines the range of the data, with 3 technical replicates shown as circles.

Fig. 3. KRAS hydrolysis based on different segments of RAF.

A Representative native mass spectra recorded at 0 min, 120 min, and 360 min for 1 µM KRAS loaded with GTP at 25°C mixed with equimolar CRAF-RBDCRD. The abundance of KRAS•GDP increases as GTP hydrolyzes. Shaded blue and red boxes denote KRAS•GDP and KRAS•GTP, respectively. Triangle denotes first species with bound sodium or magnesium adducts. B–E Percentage of hydrolyzed GDP at 360 min of 1 µM (B) KRAS, C G12C, D G13D, and E Q61H bound to equimolar (MEK1)(BRAF)(14-3-3)₂, CRAF-RBD, and CRAF-RBDCRD._. Two-tailed t-test was performed, with two asterisks denoting p values < 0.01. The error bar defines the range of the data, with 3 technical replicates shown as circles.

Fig. 4. Phosphorylation kinetics of ERK2 influenced by KRAS and mutants.

A Representative deconvoluted native mass spectra recorded at 0 min, 40 min, and 80 min incubated at 25°C with 1 µM KRAS, 1 µM (MEK1)(BRAF)(14-3-3)₂, 1 µM ERK2, 100 µM ATP, 100 µM Mg(OAc)₂. The addition of 80 and 160 Da correspond to one and two phosphorylation, respectively. Triangles denote species with bound sodium or magnesium adducts. B Same conditions and mixture as (A) supplemented with 1.25 µM GDC-0623. C The same conditions from panel A were used. The concentration of ERK with zero (green), one (blue), or two (pink) phosphorylation was determined by deconvolution of native MS data (dots) and fit to a first-order rate constant model (lines). D Rate constant for first phosphorylation (left) and second phosphorylation (right) determined by native MS. Two-tailed t-test was performed, with one asterisk denoting p-values < 0.05 and two asterisks denoting p values < 0.01. The error bar defines the range of the data, with 3 technical replicates shown as circles.