Phenotypic Mutation 'gigante' (pdf version)
Allelegigante
Mutation Type missense
Chromosome5
Coordinate117,809,514 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Ksr2
Gene Name kinase suppressor of ras 2
Chromosomal Location 117,552,067-117,906,061 bp (+) (GRCm39)
MGI Phenotype PHENOTYPE: Homozygous mice exhibit increased body fat and obesity, resulting from hyperphagia. Mice are also glucose intolerant and have high serum cholesterol, ALT, serum lipids and show hepatic steatosis. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_001114545; MGI:3610315

MappedYes 
Amino Acid Change Cysteine changed to Serine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000137670]
AlphaFold no structure available at present
SMART Domains Protein: ENSMUSP00000137670
Gene: ENSMUSG00000061578
AA Change: C426S

DomainStartEndE-ValueType
Pfam:KSR1-SAM 24 152 1.1e-45 PFAM
low complexity region 258 282 N/A INTRINSIC
low complexity region 326 341 N/A INTRINSIC
low complexity region 357 368 N/A INTRINSIC
C1 412 457 2.74e-8 SMART
low complexity region 518 551 N/A INTRINSIC
low complexity region 617 637 N/A INTRINSIC
Pfam:Pkinase 667 929 1.1e-41 PFAM
Pfam:Pkinase_Tyr 667 929 1.8e-46 PFAM
Predicted Effect probably damaging

PolyPhen 2 Score 0.994 (Sensitivity: 0.69; Specificity: 0.97)
(Using ENSMUST00000180430)
Meta Mutation Damage Score 0.9685 question?
Is this an essential gene? Probably nonessential (E-score: 0.103) question?
Phenotypic Category Autosomal Recessive
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(7) : Gene trapped(2) Targeted(5)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
IGL02136:Ksr2 APN 5 117754959 missense possibly damaging 0.52
IGL02231:Ksr2 APN 5 117638841 missense probably damaging 1.00
IGL02634:Ksr2 APN 5 117901394 splice site probably benign
IGL02669:Ksr2 APN 5 117693446 missense probably damaging 1.00
IGL03116:Ksr2 APN 5 117846022 missense probably benign 0.20
IGL03168:Ksr2 APN 5 117886846 missense probably damaging 1.00
IGL03372:Ksr2 APN 5 117840783 missense possibly damaging 0.93
float UTSW 5 117809523 missense probably damaging 1.00
loft UTSW 5 117638857 missense probably benign 0.10
R0133:Ksr2 UTSW 5 117693359 missense possibly damaging 0.95
R0811:Ksr2 UTSW 5 117693290 missense probably damaging 1.00
R0812:Ksr2 UTSW 5 117693290 missense probably damaging 1.00
R1162:Ksr2 UTSW 5 117693020 splice site probably benign
R1420:Ksr2 UTSW 5 117552904 missense probably benign 0.10
R1717:Ksr2 UTSW 5 117809514 missense probably damaging 0.99
R1809:Ksr2 UTSW 5 117693535 missense probably damaging 1.00
R1859:Ksr2 UTSW 5 117553006 missense probably damaging 1.00
R1867:Ksr2 UTSW 5 117643594 missense probably benign 0.32
R1868:Ksr2 UTSW 5 117643594 missense probably benign 0.32
R3024:Ksr2 UTSW 5 117693125 missense possibly damaging 0.52
R3499:Ksr2 UTSW 5 117827640 missense probably damaging 1.00
R3687:Ksr2 UTSW 5 117693044 missense probably damaging 0.98
R3688:Ksr2 UTSW 5 117693044 missense probably damaging 0.98
R4044:Ksr2 UTSW 5 117693127 nonsense probably null
R4579:Ksr2 UTSW 5 117894335 missense probably damaging 0.99
R4697:Ksr2 UTSW 5 117846212 missense probably damaging 1.00
R4834:Ksr2 UTSW 5 117806392 missense probably benign 0.37
R5016:Ksr2 UTSW 5 117638857 missense probably benign 0.10
R5107:Ksr2 UTSW 5 117827673 missense probably benign 0.01
R5150:Ksr2 UTSW 5 117693074 missense probably damaging 0.97
R5326:Ksr2 UTSW 5 117846305 missense probably damaging 1.00
R5493:Ksr2 UTSW 5 117846175 missense probably damaging 1.00
R5738:Ksr2 UTSW 5 117886864 missense probably damaging 0.97
R6257:Ksr2 UTSW 5 117552909 missense probably benign 0.01
R6316:Ksr2 UTSW 5 117823567 missense probably damaging 1.00
R6389:Ksr2 UTSW 5 117552907 missense probably benign 0.09
R6460:Ksr2 UTSW 5 117894449 critical splice donor site probably null
R6874:Ksr2 UTSW 5 117894401 nonsense probably null
R6939:Ksr2 UTSW 5 117903626 makesense probably null
R7352:Ksr2 UTSW 5 117827706 missense probably benign 0.00
R7594:Ksr2 UTSW 5 117693131 missense possibly damaging 0.89
R7840:Ksr2 UTSW 5 117693329 missense probably benign 0.00
R7919:Ksr2 UTSW 5 117899418 missense possibly damaging 0.86
R8152:Ksr2 UTSW 5 117809523 missense probably damaging 1.00
R8949:Ksr2 UTSW 5 117823560 missense possibly damaging 0.68
R9133:Ksr2 UTSW 5 117841319 missense probably benign 0.02
R9299:Ksr2 UTSW 5 117885399 critical splice acceptor site probably null
R9356:Ksr2 UTSW 5 117827706 missense probably benign 0.40
R9592:Ksr2 UTSW 5 117894344 missense probably damaging 1.00
R9658:Ksr2 UTSW 5 117885425 missense probably damaging 1.00
RF020:Ksr2 UTSW 5 117693283 missense probably benign
Z1088:Ksr2 UTSW 5 117885467 missense probably damaging 1.00
Z1177:Ksr2 UTSW 5 117885473 missense probably damaging 0.99
Z1177:Ksr2 UTSW 5 117846265 missense probably damaging 1.00
Mode of Inheritance Autosomal Recessive
Local Stock Live Mice, Sperm, gDNA
MMRRC Submission 038210-MU
Last Updated 2019-09-04 9:46 PM by Anne Murray
Record Created 2015-03-12 1:27 PM by Jeff SoRelle
Record Posted 2015-08-07
Phenotypic Description
Figure 1. Phenotype of the gigante mice (right). A C57BL/6J wild-type littermate (left) is shown for comparison.

The gigante phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1717, some of which increased body weights compared to wild-type mice (Figure 1).

Nature of Mutation

Whole exome HiSeq sequencing of the G1 grandsire identified 102 mutations. Among these, only one affected a gene with known effects on body weight, Ksr2. The mutation in Ksr2 was presumed to be causative because the gigante body weight phenotype mimics other known alleles of Ksr2 (see MGI for a list of Ksr2 alleles). The Ksr2 mutation is a T to A transversion at base pair 117,671,449 (v38) on chromosome 5, or base pair 257,680 in the GenBank genomic region NC_000071. The mutation corresponds to residue 2,103 in the mRNA sequence NM_001114545 within exon 7 of 21 total exons.

2088 TGGATGTCTCAGACGTGCACGGTCTGCGGGAAA

421  -W--M--S--Q--T--C--T--V--C--G--K-

The mutated nucleotide is indicated in red.  The mutation results in a cysteine (C) to serine (S) substitution at position 426 (C426S) in the kinase suppressor of Ras2 (KSR2) protein, and is strongly predicted by PolyPhen-2 to cause loss of function (score = 0.997) (1).

Illustration of Mutations in
Gene & Protein
Protein Prediction
Figure 2. KSR2 domain organization. The five conserved domains (CA1-CA5) are labeled. The schematic shows the relative positions of the Cys-rich domain (CRD), Ser/Thr-rich sequences, kinase domains, coiled-coil sterile α-motif (CC-SAM), and Pro-rich sequence. The gigante mutation results in a cysteine (C) to serine (S) substitution at position 426 (C426S) and is marked by a red asterisk.

KSR2 is a member of the RAF family of protein kinases. The KSR proteins share similar domain organization to other members of the RAF family. KSR proteins have five conserved domains termed CA1 to CA5 (Figure 2). The CA1 domain corresponds to a coiled-coil-sterile α motif (CC-SAM; amino acids 24-152), which is necessary for localization of KSR2 to the plasma membrane (2). The CA2 domain is proline-rich and has a Src homology 2 (SH2) domain; the function of the CA2 domain is unknown. The CA3 domain is a cysteine-rich C1 domain (CRD; amino acids 412-457), which regulates the translocation of the KSR proteins to the plasma membrane (3). The CA4 domain is a serine/threonine-rich domain and contains an ERK binding motif (4). Within the CA4 domain is a consensus MAPK phosphorylation site (FXFP). The CA5 domain corresponds to the conserved kinase catalytic domain (amino acids 667-929) (5). The kinase function of the KSR proteins is unclear as studies have shown both a presence (6-8), and an absence (9-11) of kinase activity. The CA5 domain also mediates the interaction between KSR and MEK1. The crystal structure of the human KSR2 kinase domain (KSR2(KD); amino acids 634-950) in complex with MEK1 has been solved [PDB:2Y4I; (6)]. The KSR2(KD)—MEK1 complex assembled into a heterotetramer through a KSR2(KD) homodimer interface mediated by Arg718. The KSR2(KD) and MEK1 proteins interact with their respective catalytic sites facing each other through their activation segments and αG helices.

Amino acids 682-690 are predicted to mediate ATP binding, a process necessary for KSR function. Mutation of Ala690 to a threonine resulted in impaired ATP binding. KSR2 has several putative phosphorylation sites throughout the length of the protein that may serve to regulate its function.

Human KSR2 has two alternatively spliced isoforms that encode protein variants that differ from full-length KSR2 within the CA1 and CA4 domains. One variant lacks the first 29 amino acids of KSR2 (hKSR2ΔN29), while the second variant lacks the entire CA1 domain (hKSR2ΔCA1). The first two exons of a third human KSR2 variant, KSR2b, are located in intron 4 of full-length hKSR2 and the last exon is within intron 14 of full-length hKSR2 resulting in a shortened CA5 domain than full-length hKSR2 (12). KSR2 variants have also been identified in the mouse, including a homolog of KSR2ΔCA1. Another variant of mouse KSR2, T-KSR2, lacks both the CA1 and CA2 domains. The first exon of T-KSR2 (exon1t) is within intron 5 of full-length KSR2, resulting in a novel 55 base pair coding sequence in the same reading frame as full-length KSR2 mRNA (12).

The gigante mutation results in a cysteine (C) to serine (S) substitution at position 426 (C426S) within the CRD (i.e., the CA3 region). The localization of KSR2gigante has not been determined.

Expression/Localization

Human KSR2 (hKSR2ΔCA1) is mainly expressed in the brain and kidney (5). Mouse Ksr2 was detected at high levels in the brain as well as at very low levels in skeletal muscle, liver, and adipose tissue (12;13). KSR2 protein was detected in mouse brain, pancreas, and purified T and B cells from the spleen (13-15). The mouse homolog of hKSR2ΔCA1 was detected in the kidney (5). T-KSR2 is specific to testes and mature sperm (12). KSR2 is a cytoplasmic protein.

Background
Figure 3. KSR proteins within the RAS-ERK pathway. (A) The RAS-ERK signaling pathway. Core components of a typical RAS–ERK signalling cascade are depicted. (1) Incoming signals from ligand-activated receptor Tyr kinases (RTKs) activate RAS. (2) Activated RAF phosphorylates MEK, which in turn (3) activates ERK. Upon activation, ERK phosphorylates a wide range of targets that can elicit various cellular responses, including growth, proliferation, differentiation, survival and migration. (B) The Raf activation cycle. Steps involved in KSR regulation often parallel those defined for RAF proteins. Step 1: Inactive KSR proteins are kept in the cytosol through interaction with inhibitory 14-3-3 proteins. KSR and MEK proteins form constitutive complexes. Step 2: the dephosphorylation of Ser406 allows 14-3-3 release and plasma membrane anchoring of KSR proteins via conserved area 1 (CA1) and CA3. Step 3: KSR proteins heterodimerize with other RAF proteins, leading to RAF transactivation and MEK–ERK signalling. Step 4: ERK-mediated negative feedback phosphorylation of several sites in RAF and KSR disrupts RAF–KSR dimers, leading to signal attenuation. Figure and legend adapted from Lavoie, H. and Therrien, M. Nat. Rev. Mol. Cell Biol. (2015), 16:281-298.

Downstream of activated receptor tyrosine kinases, RAS is activated in the RAS/RAF/MEK/ERK signaling pathway. RAF and the KSR proteins are subsequently regulated by the activated RAS. Upon activation, RAF phosphorylates MEK, which phosphorylates ERK1/2 (see the record wabasha). KSR proteins (KSR2 and KSR1) function as scaffold proteins and mediate the spatiotemporal regulation of the RAS/RAF/MEK/ERK signaling pathway (4;16;17). Inactive KSR proteins are kept in the cytosol through an interaction with inhibitory 14-3-3 proteins [reviewed in (18)]. Dephosphorylation of 14-3-3 facilitates its release and the subsequent plasma membrane anchoring of the KSR proteins via the CA1 and CA3 domains. KSR assembles into a MEK-KSR-RAF complex to promote RAF-mediated phosphorylation of MEK (6;11;19;20). The KSR proteins heterodimerize with RAF proteins, promoting RAF transactivation and subsequent MEK-ERK signaling. Activated ERK1/2 phosphorylates several targets, which subsequently are involved in several processes including cell cycle progression, cell migration, adhesion, survival, differentiation, metabolism, proliferation, and transcription. ERK-mediated phosphorylation of RAF and KSR results in the dissociation of RAF and KSR and attenuation of signaling.

KSR2 also binds AMP-activated protein kinase (AMPK) to promote signal transduction (13;15;16). AMPK is a primary regulator of cellular energy homeostasis. In response to a metabolic need, AMPK stimulates energy (ATP) production (e.g. glucose and lipid catabolism) or inhibits energy consumption (e.g. anabolic pathways including protein, fatty acid, and cholesterol synthesis) (21). AMPK activation occurs upon increased AMP concentration and by phosphorylation of the α subunit (Thr172) by serine/threonine kinase 11 (LKB1), Ca2+/ calmodulin-dependent protein kinase kinase β (CaMKKβ), and transforming growth factor-β-activated kinase (TAK1) (21;22). AMPK activation downregulates functional NF-κB signaling through downstream mediators (e.g. Sirtuin 1 (SIRT1), the Forkheadbox O (FoxO) family, and peroxisome proliferator-activated receptor-activated receptor γ co-activator 1α (PGC-1α)) (21;23;24).  AMPK activation results in glucose and fatty acid uptake, glycolysis, and fatty acid oxidation as well as in the enhancement of mitochondrial biogenesis, the inhibition of fatty acid synthesis, gluconeogenesis, glycogen storage, and cholesterol biosynthesis (25). Decreased AMPK activation in Ksr2-deficient (Ksr2-/-) mice resulted in impaired oxidation of fatty acids and a subsequent increase in their storage as triglycerides, leading to obesity and insulin resistance (13). Some KSR2 mutations in humans result in early onset obesity, and are due to disrupted ERK activation or disrupted interaction between KSR2 and AMPK (26).

The hKSR2ΔCA1 KSR2 variant is a regulator of Cot/TPL2-induced MAPK signaling. Cot (see the record for Sluggish) is a serine/threonine kinase of the MAP kinase kinase kinase (MAP3K) family that is an upstream factor in the ERK signaling pathway (5). Under basal conditions, TPL2 binds to NF-κB1 p105 (see the record for Finlay) and the A20 binding inhibitor of NF-kappaB activation (ABIN)-2, where it exists in a stable but inactive state (27-29). Upon toll-like receptor (TLR) stimulation, both p105 and TPL2 are phosphorylated by the IKK complex, resulting in degradation of p105 and the release and activation of TPL2 (30). Phosphorylation of TPL2 by the IKK complex is necessary for both the dissociation of TPL2 from p105 and TPL2 kinase activity (31;32).  Activated TPL2 phosphorylates MEK1/2 (MAP kinase 1 and 2), which then activates ERK1/2 (27). Interaction between hKSR2ΔCA1 and Cot attenuates Cot-mediated ERK and NF-κB activation (5)

KSR2 regulates IL-1β-induced, MEKK3-mediated NF-κB activation by regulating MAPK kinase kinase 3 (MEKK3; alternatively, MAP3K3) (33). The serine/threonine kinase MEKK3 activates the ERK, JNK, and p38 signaling pathways in response to several stimuli including cellular stress and inflammatory cytokines, resulting in cell proliferation, differentiation, apoptosis, and inflammatory responses.  For example, stimulation of TLR4 (see the record for lps3) by lipopolysaccharide activates both the MyD88-dependent and MyD88-independent signaling pathways. In the MyD88-dependent pathway, MyD88 is recruited to the receptor, where it functions as an adapter to recruit IRAK family proteins, first IRAK-4 and then IRAK-1, as well as TRAF6. The ensuing signaling pathway culminates in the activation of NF-κB-dependent transcription. Briefly, IRAK-1 and TRAF6 dissociate from the receptor complex, and freed TRAF6 interacts with, and activates, the MAP3Ks TAK1 and MEKK. Activated TAK1 and MEKK3 subsequently phosphorylate the IκB kinase (IKK) complex. The IKK complex phosphorylates IκB, targeting it for degradation and relieving its inhibition of NF-κB which translocates to the nucleus and activates expression of target genes including interleukin (IL)-6, IL-1, TNF, IL-12p40 and type I interferon, cytokines required for the inflammatory response.

KSR2 is essential for optimal calcium entry during store-operated calcium entry (SOCE) in both T- and B-lymphocytes (14). SOCE is the primary method of calcium entry in nonexcitable cells. In resting T cells, a calcium gradient exists between the cytoplasm and the extracellular space. Upon antigen recognition through the T cell receptor as well as binding of chemokine receptors, activated phospholipase β (PLCβ) and PLCγ1 (see the record queen for information on PLCγ2) catalyze the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) to inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) [reviewed in (34)]. InsP3 binds to and opens InsP3 receptors (InsP3Rs) in the membrane of the ER, resulting in the release of Ca2+ from intracellular Ca2+ stores. A decrease in the Ca2+ content of the ER is 'sensed' by stromal interaction molecule 1 (STIM1), which in turn activates calcium-release-activated calcium (CRAC) channels in the plasma membrane. Increased intracellular Ca2+ levels activate enzymes, including the protein phosphatase calcineurin, CaMKII, and IKK. KSR2 functions in calcium-mediated ERK signaling through an interaction with calcineurin (15). In response to calcium signals, calcineurin regulates KSR2 localization and ERK scaffold activity as well as the de-phosphorylation, and activation, of the nuclear factor of activated T-cell (NFAT) transcription factor within the cytoplasm (15). Activation of NFAT promotes its translocation into the nucleus, where it activates the transcription of immune response genes in T lymphocytes as well as other genes in several diverse cell types.

Mutations in KSR2 has been linked to obesity and insulin resistance in humans  as well as increased energy intake in children (26). Fasting insulin levels were higher and glucose tolerance impaired in individuals with KSR2 mutations. A link between KSR2 expression and tumor cell growth has also been proposed. KSR2 expression increased the proliferation of mouse embryonic fibroblasts (MEFs), while loss of KSR2 expression reduced tumor cell growth (35). In addition, KSR2 promoted anchorage-independent growth (35). RNA interfence (RNAi)-induced knockdown of KSR2 expression in MIN6 and NG108-15 tumor cells resulted in reduced proliferation and colony formation with a concomitant reduction in AMPK signaling, nutrient metabolism, and metabolic capacity (35).

Putative Mechanism

Ksr2-deficient (Ksr2-/-) mice are obese by approximately 9-10 weeks of age and exhibit insulin resistance (13;36-38); Ksr2-/- mice are comparable in weight and size to wild-type mice at the time of weaning (13;36;38). The Ksr2-/- mice ate more food and gained more weight than wild-type mice (36). When food intake was controlled, the Ksr2-/- mice gained more weight than wild-type mice due to an increase in fat mass, indicating reduced energy expenditure in the Ksr2-/- mice compared to wild-type mice (36;38). Costanzo-Garvey et al. observed a lower metabolic rate in Ksr2-/- mice compared to wild-type mice; the locomotor activity of the Ksr2-/- mice was comparable to wild-type mice (13). The Ksr2-/- mice had high levels of hemoglobin A1c and fasted serum glucose, and low levels of adiponectin (36). Henry et al. determined that the Ksr2-/- mice were not born with defective glucose regulation, but that defects develop as a result of obesity (38). Food restrictions after weaning of Ksr2-/- mice prevented defective glucose homeostasis. In addition, by restricting the diet of adult Ksr2-/- mice, glucose homeostasis could be restored (38). Ksr2-/- mice fed low-fat diet had high serum total cholesterol levels and higher triglyceride levels than wild-type mice (36). After a high-fat diet, the Ksr2-/- mice had higher levels of serum lipids and liver weights than wild-type mice (36). Both male and female Ksr2-/- mice exhibit impaired fertility (13). Ksr2-/- female mice have impaired mammary development as well as they begin estrous cycles later than wild-type mice. Ksr2-/- male mice exhibit reduced sex drive and copulate infrequently. The Ksr2-/- mice exhibited decreased viability and approximately 50% of the mice were dead by 16 weeks of age (36). The Ksr2-/- mice had hypophagia, weight loss, inactivity, and hypothermia in the last 1-2 weeks of life. Similar to the Ksr2-/- mice, the gigante mice exhibited higher body weights than wild-type littermates, indicating a loss of KSR2gigante function.

Primers PCR Primer
gigante_pcr_F: AGCAACTTGATGAAACTTAGTTGCC
gigante_pcr_R: GGCTTTCCTCAGAGCTGTTC

Sequencing Primer
gigante_seq_F: AGTTGCCACATGTCTTGGAAC
gigante_seq_R: TGCCTGGGACCTTTCTAAAATAC
Genotyping

PCR program

1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40x
6) 72°C 10:00
7) 4°C hold


The following sequence of 437 nucleotides is amplified (chromosome 5, + strand):


1   agcaacttga tgaaacttag ttgccacatg tcttggaaca aacaaggctg gcatccagag
61  catatcagct tcccatcctc ggggtgcatc ctgctgactg tgtggacagt gactcacctt
121 ggacatttcc tgtttctttc ccacaggttt tccaccaagt actggatgtc tcagacgtgc
181 acggtctgcg ggaaagggat gctttttggc ctcaagtgta aaaactgcaa gtgagtgctt
241 tttctttaag ggacttttta caatgccaga tgccagccag tgtgggggat gggcatgggc
301 atggtccagc aagtgtgagc tcttgactct gggagagatg attttttttt tttttttttt
361 tggctttgta ttttagaaag gtcccaggca gagctgtgtg cttgaggaga gtatcaagaa
421 cagctctgag gaaagcc


Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red.

References
Science Writers Anne Murray
Illustrators Peter Jurek
AuthorsJeff SoRelle