For over 3 decades, the biochemical effects exhibited by cancer-associated RAS mutations have been studied in great detail. In contrast, only limited information is available on the newly discovered germline mutations of RAS. The data presented herein represent the most comprehensive biochemical and structural analysis of these novel RAS mutants to date.
The key phenomenon in RAS biology is its nucleotide-dependent interaction with different proteins of the signal transduction machinery, which is controlled by the GDP/GTP exchange and the GTP hydrolysis reactions. Any change of these functions or an impairment of the interaction of RAS with its binding partners can affect the fine-tuned balance of RAS regulation and its activity in cells. It is now clear that aberrant RAS function in the developing embryo leads to an abnormal progression of developmental programs. Understanding the mechanisms by which aberrant RAS disturbs normal development represents an important scientific goal. The results of this study confirm that germline KRAS mutations generally confer a milder gain-of-function phenotype than cancer-associated mutations at positions G12, G13, or Q61. Moreover, we show that germline KRAS mutations caused multifaceted effects that cannot simply be explained to result from one of the underlying mechanisms fine-tuning RAS functions. To gain insights into the structural alterations caused by the germline KRAS mutations, we inspected the environment of the respective residue and compared them with RASwt to explain their functional properties (Supp. Fig. S7, and “Assessement of possible structural consequences of RAS mutants” in the online Supporting Information). As summarized below and in Table 1 and Supp. Table S4, our data strongly suggest the existence of five partially interrelated mechanistic classes of KRAS mutants with altered signal transduction:
Class A groups the mutants KRASK5N, KRAST58I, and KRASD153V, which do not show major biochemical alterations compared to wild-type KRAS in vitro. All three mutants, especially p.T58I, are in a higher activated state and show a higher downstream signaling compared to RASwt indicating that mutation at these positions do impact RAS function but could not be monitored by the current tools of RAS biochemistry. KRASD153V expressing cells showed a slightly higher GTP-bound level and an increase in MEK1/2 phosphorylation compared to KRASwt but no difference regarding pERK1/2 and pAKT levels. The reason for this observation is not understood yet.
On the other hand, we have recently shown in a similar situation with NRAST50I
identified in NS patients, that the residue T50 does not play a functional role, either in nucleotide binding and hydrolysis or in contacting protein partners of NRAS, but rather in the interaction with membrane lipids [Cirstea et al., 2010
]. By inspecting such a RAS/membrane model [Abankwa et al., 2008
], it is rather tempting to speculate that the E153 (in HRAS)/D153 (in KRAS) side chain may directly influence RAS interaction with the membrane. Moreover, we superposed HRAS with a recently published KRAS mutant structure (Supp. Table S1), which clearly showed that there is no significant difference between the E153 and D153 positions (Supp. Fig. S6).
Class B represented by KRASV14I
, showed a dramatic increase, both in intrinsic and GEF-catalyzed nucleotide exchange as the probable major cause for its accumulation in the GTP-bound state and increased downstream signaling. In contrast to a previous study, where KRASV14I
exhibited a significantly lower GTP-hydrolysis in the presence of RAS-specific GAPs in vitro [Schubbert et al., 2006
], we did not observe any changes in the intrinsic and GAP-stimulated GTP-hydrolysis reactions () and an only mild decrease in effector binding affinity.
Class C is represented by KRASQ22R
, and characterized by an impaired GAP-stimulated GTP hydrolysis while its intrinsic functions including the intrinsic GTP hydrolysis reaction () remained unaffected and its interaction with effectors is virtually functional (). Consistent with our results, a KRASQ22K
mutant, which is physiologically homologous to p.Q22R, has been shown to transform NIH-3T3 fibroblasts [Tsukuda et al., 2000
], an effect that is presumably caused by accumulation of RAS in its active state. The underlying pathogenetic mechanism is most likely due to a surface exposed guanidinium group of the arginine which prevents GAP binding (Supp. Fig. S7C) but does not interfere with effector binding ().
Class D comprises the mutants KRASQ22E
. The members of this class are characterized by an increase in intrinsic and catalyzed nucleotide exchange in combination with the resistance to GAPs, but still with a functional interaction with effectors. These effects which cause a profound activation of the MAPK and PI3K/AKT pathways are not directly affecting nucleotide binding and hydrolysis ( and ) because Q22 and F156 are not directly involved in the coordination of the active center. F156 substitution by leucine creates a cavity within the hydrophobic core causing loss of contact with surrounding residues (Supp. Fig. S7E), which lead to an overall reduction of the nucleotide binding affinity [Xu et al., 1998
], an increase in the cellular level of GTP-bound RAS and subsequent activation of the transforming potential of RAS [Quilliam et al., 1995
All mutations that cause faster nucleotide dissociation (KRASV14I, KRASQ22E, KRASF156L) in comparison to RASwt affect amino acids that are either barely (V14, Q22) or not at all (F156) exposed to the solvent ( and Supp. Table S3). It implicates that disturbed integrity of RAS structure is responsible for the alteration of its intrinsic property as the substitutions of buried amino acids by smaller side chains very likely affect the internal dynamics of the proteins.
Class E is represented by the mutants KRASP34L
, and KRASG60R
and is characterized by a defective GAP sensitivity and a strongly reduced interaction with effectors. Although these mutants are locked in a hyperactivated state, which is rather comparable to the oncogenic RASG12V
, their ineffectiveness for downstream signaling in turn causes only a mild gain-of-function phenotype. Accordingly, class E mutants are able to activate downstream pathways as shown by ERK1/2 and AKT phosphorylation (). A similar case has been recently reported in a germline HRAS mutant associated with CS [Gremer et al., 2010
]. Hereby, E37 duplication in the switch I region of HRAS impairs both binding of GAP and effector proteins. Therefore, this mutant can also be assigned as a class E member. Although KRASP34L
do not respond to GAP they are in principle able to hydrolyze GTP intrinsically (). This strongly suggests that the respective amino acid substitutions either interfere with GAP binding or with the positioning of the catalytic arginine of GAP in the active site. P34 is invariant in RAS and RHO proteins [Eberth et al., 2005
] and any substitution of P34 has been suggested to affect interaction with the binding partner of RAS [Chung et al., 1993
; Stone et al., 1993
The mutation of KRAS G60 to arginine has most severe consequences, namely, an overall impairment of almost all biochemical and functional properties. Its substitution by a large and charged amino acid like arginine in KRAS or glutamate in NRAS [Cirstea et al., 2010
] seems to corrupt the switch regions including the critical catalytic Q61, affect nucleotide binding, GTP hydrolysis, and impairs intermolecular interaction with regulators and effectors. Previous studies have shown that a conservative mutation of G60 to alanine impairs the normal GTPase function of RAS and Gα [Ford et al., 2005
; Sung et al., 1996
]. G60A mutation of HRAS dramatically affects intrinsic and GAP-stimulated GTP hydrolysis without major changes on its interaction with effector proteins [Hwang et al., 1996
]. Structural analysis of HRASG60A
showed that its switch I region adopts an open conformation [Ford et al., 2005
]. However, G60 substitution by arginine (KRAS) or glutamate (NRAS) may affect both switch regions, which in turn may be the reason for loss of intrinsic and extrinsic functions.
Interestingly, certain RAS mutations such as p.P34L, p.P34R, or p.G60R are compromised in their interaction with effectors as evidenced by the inability to bind efficiently RAF1–RBD or RALGDS–RBD ( and Supp. Fig. S5). This is surprising considering that enhanced downstream signaling is the primary cause of the developmental diseases. At the same time, these mutations have the most severe effect on the GAP-mediated GTPase reaction, in a range that is quite similar to the oncogenic mutation prototype p.G12V. It is likely that the impairment of effector interactions damps the consequences of GAP resistance of these mutants on downstream signal flow. This is in contrast to RAS proteins with oncogenic mutations at the positions G12 or Q61, which contribute to potent transforming properties [Der et al., 1884; Seeburg et al., 1886]. Our results therefore provide an explanation for the lower levels of activated KRAS signaling exerted by germline mutations compared to the classical oncogenic mutations. The fact that the majority of investigated amino acids of RAS are neither involved in contacts with interacting partners nor with the nucleotide also suggests that the effects of changes at these sites are milder compared to oncogenic mutations and may at least in part explain why these alterations are tolerated in the germline and are generally not associated with tumor development in affected individuals [Karnoub and Weinberg, 2008
; Quinlan and Settleman, 2009
The diversity of functional consequences of germline KRAS
mutations is paralleled by a remarkably wide phenotypic spectrum associated with mutations in this gene and its tempting to assume a causal relation between certain genotypes and phenotypic expressions. There is indeed a tendency towards an association of more severe phenotypes (CFC/CS) with mutations that proved to have stronger effects on ERK1/2 phosphorylation in our experiments (p.Q22E, p.Q22R, p.P34R, p.G60R, p.F156L) (). In contrast, patients harboring the mutations p.V14I, p.P34L, p.D153V tend to have less severe physical and mental handicaps and are more commonly classified as having NS, the less severe form among this group of developmental disorders [Aoki et al., 2008
]. However, the number of known patients with a proven KRAS
mutation is still too small to delineate clear genotype–-phenotype correlations.
In conclusion, we describe and classify in detail the functional properties of a spectrum of germline mutations of KRAS that have been previously identified as a cause of developmental syndromes. Our studies reveal several new mechanisms by which germline KRAS mutations contribute to human disease and lead to disturbed embryonic development.