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, 4 (1), 71-80

Comparative Nucleotide-Dependent Interactome Analysis Reveals Shared and Differential Properties of KRas4a and KRas4b

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Comparative Nucleotide-Dependent Interactome Analysis Reveals Shared and Differential Properties of KRas4a and KRas4b

Xiaoyu Zhang et al. ACS Cent Sci.

Abstract

The KRAS gene encodes two isoforms, KRas4a and KRas4b. Differences in the signaling functions of the two KRas proteins are poorly understood. Here we report the comparative and nucleotide-dependent interactomes of KRas4a and KRas4b. Many previously unknown interacting proteins were identified, with some interacting with both isoforms while others prefer only one. For example, v-ATPase a2 and eIF2Bδ interact with only KRas4b. Consistent with the v-ATPase interaction, KRas4b has a significant lysosomal localization. Comparing WT and constitutively active G12D mutant KRas, we examined differences in the effector proteins of the KRas4a and KRas4b. Interestingly, KRas4a binds RAF1 stronger than KRas4b. Correspondingly, KRas4a can better promote ERK phosphorylation and anchorage-independent growth than KRas4b. The interactome data represent a useful resource to understand the differences between KRas4a and KRas4b and to discover new function or regulation for them. A similar proteomic approach would be useful for studying numerous other small GTPases.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Identifying KRas4a and KRas4b interacting proteins in HEK293T cells by SILAC and AP-MS. (a) Scheme showing identification of KRas4a/b interacting proteins in HEK293T cells by SILAC and AP-MS. (b) Plotting KRas4a/b interacting proteins (with ≥2 unique peptides) against their heavy/light ratios. (c) Nonspecific versus confident interacting proteins of KRas4a/b identified in HEK293T cells. (d) Heat map showing the heavy/light ratios of KRas4a/b interacting proteins in HEK293T cells. (e) iBAQ values (from HEK293T cells) showing the abundance distribution of KRas4a/b interacting proteins. (f) Heavy/light ratio and peptide number (within parentheses) of known KRas4a/b interacting proteins. (g) Venn diagrams showing the numbers of shared and unique interacting proteins of KRas4a/b in HEK293T cells. (h) Biological process analysis of KRas4a/b interacting proteins. Categories were assigned based on DAVID analysis UP_KEYWORDS.
Figure 2
Figure 2
KRas4b interacts with v-ATPase a2 through its C-terminal HVR. (a) Heavy/light ratio of v-ATPase a2 in KRas4a/b SILAC and the primary mass spectra of one v-ATPase a2 peptide (residues 638–650) in forward and reverse KRas4b SILAC. (b) Immunoprecipitation of FLAG-tagged KRas4b, but not KRas4a, pulled out endogenous v-ATPase a2 in HEK293T cells. (c) Immunoprecipitation of FLAG-tagged HRas(1–164)-KRas4b (165–188) (labeled as FLAG-HRas+K4b), but not FLAG-tagged HRas(1–164)-KRas4a (165–189) (labeled FLAG-HRas+K4a), pulled out endogenous v-ATPase a2 as FLAG-tagged KRas4b did. (d) Immunoprecipitation of both FLAG-tagged KRas4b G12D and S17N pulled out similar levels of endogenous v-ATPase a2. RAF1 was used as a positive control, which only interacted with KRas4b G12D but not S17N. (e) Confocal images showing the colocalization of GFP-KRas4a WT or GFP-KRas4b WT with LAMP1 in HEK293T cells. Quantification of colocalization was shown on the left using Pearson’s coefficient (n = 10 for each sample). Statistical evaluation was done using a two-way ANOVA. Scale bar: 5 μm.
Figure 3
Figure 3
KRas4b interacts with eIF2Bδ through its C-terminal HVR. (a) Heavy/light ratio of eIF2Bδ in KRas4a/b SILAC and the primary mass spectra of one eIF2Bδ peptide (residues 210–225) in forward and reverse KRas4b SILAC. (b) Immunoprecipitation of FLAG-tagged KRas4b, but not KRas4a, pulled out endogenous eIF2Bδ in HEK293T cells. (c) Immunoprecipitation of FLAG-tagged HRas(1–164)-KRas4b (165–188), but not HRas(1–164)-KRas4a (165–189), pulled out endogenous eIF2Bδ as FLAG-tagged KRas4b did. (d) Immunoprecipitation of FLAG-tagged KRas4b WT, G12D and S17N pulled out similar levels of endogenous eIF2Bδ. (e) Heavy/light ratios of eIF2Bα, eIF2Bβ, eIF2Bγ, and eIF2Bε in KRas4a/b SILAC.
Figure 4
Figure 4
KRas4a has more RAF1 interaction than KRas4b in cells. (a) Detection of interactions between endogenous RAF1 and FLAG-tagged WT and G12D mutants of HRas, NRas, KRas4a, and KRas4b in HEK293T cells. (b) FLAG-tagged KRas4a/b G12D and endogenous Ras expression levels in NIH 3T3 cells. FLAG-tagged KRas4a/b had higher molecular weight than endogenous Ras and ran higher on the gel. (c) Immunoprecipitation of FLAG-tagged KRas4a G12D pulled out more endogenous RAF1, but not ARAF and BRAF, than FLAG-tagged HRas G12D and KRas4b G12D did in NIH 3T3 cells. (d) Immunoprecipitation of FLAG-tagged HRas(1–164)-KRas4a(165–189) pulled out more endogenous RAF1 than FLAG-tagged HRas(1–164)-KRas4b(165–188) did in HEK293T cells. (e) p-ERK (Thr202, Tyr204) and ERK levels in NIH 3T3 cells expressing pCDH vector, FLAG-tagged HRas G12D, KRas4a G12D, or KRas4b G12D. (f) FLAG-tagged HRas G12D, KRas4a G12D, and KRas4b G12D increased the phosphorylation levels of several key proteins (p-Akt Thr308, p-Akt Ser473, p-S6K Thr389, and p-4E-BP1 Thr37,46) in the PI3K-Akt-mTOR pathway to similar extents in NIH 3T3 cells. (g) Normal 2D cell proliferation of NIH 3T3 cells expressing pCDH vector, FLAG-tagged HRas G12D, KRas4a G12D, or KRas4b G12D. Statistical evaluation was examined using an unpaired two-tailed Student t test. Error bars represent SD in three biological replicates. (h) Anchorage-independent soft agar assay showing that KRas4a G12D expressing NIH 3T3 cells had higher colony number than HRas G12D or KRas4b G12D expressing NIH 3T3 cells. Statistical evaluation was examined using an unpaired two-tailed Student t test. Error bars represent SD in three biological replicates. ***P < 0.001. (i) Knocking down RAF1 by two different shRNAs dramatically decreased KRas4a G12D and KRas4b G12D induced colony formation in soft agar assay. Top figure shows the Western blot of endogenous RAF1 in empty vector, KRas4a G12D, or KRas4b G12D expressing NIH 3T3 cells and the quantification of bands on the Western blot membrane. Statistical evaluation was examined using an unpaired two-tailed Student t test. Error bars represent SD in three biological replicates. ***P < 0.001. (j) Scheme showing that in NIH 3T3 cells, increased KRas4a-RAF1 interaction may contribute to increased anchorage-independent cell growth.

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