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. 2012 Dec 18;5(255):ra94.
doi: 10.1126/scisignal.2003289.

A CC-SAM, for coiled coil-sterile α motif, domain targets the scaffold KSR-1 to specific sites in the plasma membrane

Dorothy Koveal  1 , Natasha Schuh-Nuhfer, Daniel Ritt, Rebecca Page, Deborah K Morrison, Wolfgang Peti
Affiliations

A CC-SAM, for coiled coil-sterile α motif, domain targets the scaffold KSR-1 to specific sites in the plasma membrane

Dorothy Koveal et al. Sci Signal. .

Abstract

Kinase suppressor of Ras-1 (KSR-1) is an essential scaffolding protein that coordinates the assembly of the mitogen-activated protein kinase (MAPK) module, consisting of the MAPK kinase kinase Raf, the MAPK kinase MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and the MAPK ERK (extracellular signal-regulated kinase) to facilitate activation of MEK and thus ERK. Although KSR-1 is targeted to the cell membrane in part by its atypical C1 domain, which binds to phospholipids, other domains may be involved. We identified another domain in KSR-1 that we termed CC-SAM, which is composed of a coiled coil (CC) and a sterile α motif (SAM). The CC-SAM domain targeted KSR-1 to specific signaling sites at the plasma membrane in growth factor-treated cells, and it bound directly to various micelles and bicelles in vitro, indicating that the CC-SAM functioned as a membrane-binding module. By combining nuclear magnetic resonance spectroscopy and experiments in cultured cells, we found that membrane binding was mediated by helix α3 of the CC motif and that mutating residues in α3 abolished targeting of KSR-1 to the plasma membrane. Thus, in addition to the atypical C1 domain, the CC-SAM domain is required to target KSR-1 to the plasma membrane.

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Conflict of interest statement

Competing interest: The authors declare that they have no competing financial interests.

Figures

Fig. 1
Fig. 1
Regions of KSR-1 are conserved across species. (A) Sequence alignment showing conservation of KSR-1 CC (α1 to α4 in blue) and SAM (α5 to α10 in green). Black and gray highlighting indicates sequence identity and similarity, respectively. The 10 α helices are depicted as cylinders above the sequence alignment. Open stars indicate residues that are buried in the individual hydrophobic cores of the CC and SAM domains, whereas filled stars indicate hydrophobic residues that are buried between them. (B) Domain architecture of KSR-1 and constructs used in this study. Conserved areas CA1 to CA5 are indicated. The CC and SAM domains do not exactly correspond to the CA1 and CA1α regions.
Fig. 2
Fig. 2
CC-SAM targets KSR-1 to the plasma membrane. (A) Serum-starved ksr-1−/− MEFs stably expressing the indicated KSR-1 proteins were stimulated with EGF, and the localization of the KSR-1 proteins was determined by immunofluorescence staining of at least 200 cells for each construct. Arrows indicate membrane ruffles. The graph on the right shows quantification of three independent localization experiments. Error bars are the SD from the mean. WT, wild type. (B) ksr-1−/− MEFs stably expressing the indicated KSR-1 proteins were serum-starved and then treated with EGF. Pyo–KSR-1 or GFP–CC-SAM proteins were immunoprecipitated and examined for binding of endogenous B-Raf and MEK proteins. The graph on the right shows densitometry performed on three immunoblots. Error bars are the SD from the mean. (C) ksr-1−/− MEFs stably expressing the GFP–CC-SAM protein were stimulated with EGF, and the localization of the GFP–CC-SAM (green) and endogenous B-Raf (red) was determined by immunofluorescence staining (experiments were repeated twice). The merged image shows the colocalization of GFP–CC-SAM and B-Raf in membrane ruffles.
Fig. 3
Fig. 3
CC interactions with SAM are mediated by hydrophobic residues. (A) Bundle of the 10 lowest energy structures of the KSR-1 CC-SAM domain. The superposition is the best fit of structured regions (residues 32 to 39, 44 to 58, 65 to 83, 87 to 91, 99 to 103, 107 to 114, 118 to 123, 130 to 135, 138 to 148, and 152 to 167 in teal; loops in gray). (B) Lowest energy structure shown as a cartoon (CC, lavender; SAM, green). Left, locations of residues discussed in the text illustrated as sticks. Right, the structure is rotated 180°, and the 10 α helices and the N and C termini are labeled. (C) Hydrophobic residues (yellow sticks) that form the interface between α2 and α3 of the CC. (D) Residues (yellow sticks) that form the hydrophobic core of the SAM region. A 90° rotation of the image appears on the right. (E) Surface representation of the CC-SAM domain, highlighting the interface (dark blue) buried between the CC (lavender) and SAM (green). (F) Thermal denaturation of the CC-SAM domain monitored using CD at 222 nm. The inset depicts the CD spectrum of the CC-SAM domain before thermal denaturation. The error bars in both plots represent the SD from the mean of three independent experiments. (G) Two hundred fifty unambiguous NOE restraints (blue lines) between the CC and SAM.
Fig. 4
Fig. 4
CC-SAM is a membrane-binding domain. (A to F) 2D [1H,15N] HSQC spectra recorded at 308 K and 500 MHz field strength of (A) 2 mM 15N CC-SAM, (B) 0.55 mM 15N CC-SAM with 82 mM SDS, (C) 0.1 mM 15N CC-SAM with 120 mM LMPG, (D) 0.1 mM 15N CC-SAM with 80 mM LPPG, (E) 0.25 mM 15N CC-SAM with 10 mM DMPG and 30 mM DHPC, and (F) 0.1 mM 15N CC-SAM with 200 mM DHPC.
Fig. 5
Fig. 5
CC-SAM domain binds directly to LMPG micelles. (A) Fully annotated 2D [1H,15N] TROSY spectrum of KSR-1 CC-SAM domain in the presence of LMPG micelles. Assigned residues are labeled with the residue name (single-letter code) and number in the sequence. The two asterisks near 10 parts per million (ppm) in the 1H dimension denote Nε1/Hε1 resonances from tryptophan side chains. (B) SSP scores of the free (blue) and micelle-bound (black) CC-SAM domain. Across the top are diagrams of the determined secondary structural elements of free and micelle-bound CC-SAM. In the micelle-bound form, dotted lines around helices α4, α5, and α7 indicate that these helices had the largest loss of helical content upon binding to micelles. (C) Of all the helices in the KSR-1 CC-SAM domain, helix α3 was most tightly associated with lipid micelles. Spectra for 2D [1H,15N] HSQC (green, 500 MHz 1H Lamor frequency) and 2D [1H,15N] TROSY (blue, 800 MHz 1H Lamor frequency) are shown. Fourteen peaks, all from helix α3, were detected only in the TROSY spectrum.
Fig. 6
Fig. 6
CC-SAM residues Ile71 and Leu78 mediate membrane binding. (A) Residues in α3 were tightly associated with micelles, as monitored by Mn2+ protection. Normalized 2D [1H,15N] HSQC peak intensities in the presence of MnCl2 (I[+Mn]) relative to those obtained in the absence of MnCl2 (I[−Mn]) are plotted for each residue. Peakswith ratios closest to 1 were most protected from the line-broadening effects of Mn2+. (B) ksr-1−/− MEFs stably expressing the indicated KSR-1 proteins were serum-starved and treated with EGF. Pyo–KSR-1 proteins were immunoprecipitated and examined for binding of endogenous B-Raf and MEK proteins. A representative blot from three separate experiments is shown. (C) Quantification of the B-Raf pull-downs is shown in (B). Error bars are the SD of the mean. (D) Serum-starved ksr-1−/− MEFs stably expressing the indicated KSR-1 proteins were stimulated with EGF, and the localization of the KSR-1 proteins was determined by immunofluorescence staining. Staining was performed in three independent experiments, and at least 200 cells were examined per experiment for each KSR-1 construct. Arrows indicate membrane ruffles. (E) Quantification of the localization results is shown in (D). Error bars are the SD of the average.
Fig. 7
Fig. 7
Targeting KSR-1 to the plasma membrane ruffles in activated cells requires both the CC-SAM and C1 domains. KSR translocates from the cytosol to the plasma membrane upon RTK activation. CC-SAM, together with the atypical C1 domain (CA3), localizes KSR-1 to B-Raf–rich microdomains in the plasma membrane. CC-SAM membrane binding is mediated by CC helix α3. Residues Ile71 and Leu78 in helix α3 are buried at the interface between helix α2 and helix α3. In the membrane-bound form of the CC-SAM, a structural rearrangement occurs whereby helix α2 releases helix α3, exposing Ile71 and Leu78, so that they can engage in membrane binding. Arg72, Lys77, and Lys80 are positioned such that they compensate for the negatively charged surface of the plasma membrane.
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