Phenotypic Mutation 'Almonde' (pdf version)
AlleleAlmonde
Mutation Type missense
Chromosome7
Coordinate122,181,672 bp (GRCm39)
Base Change T ⇒ A (forward strand)
Gene Prkcb
Gene Name protein kinase C, beta
Synonym(s) Prkcb1, A130082F03Rik, Prkcb2, Pkcb, PKC-Beta
Chromosomal Location 121,888,327-122,233,625 bp (+) (GRCm39)
MGI Phenotype FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This protein kinase has been reported to be involved in many different cellular functions, such as B cell activation, apoptosis induction, endothelial cell proliferation, and intestinal sugar absorption. Studies in mice also suggest that this kinase may also regulate neuronal functions and correlate fear-induced conflict behavior after stress. Alternatively spliced transcript variants encoding distinct isoforms have been reported. [provided by RefSeq, Jul 2008]
PHENOTYPE: Mice homozygous for a null allele exhibit impaired humoral immune responses, altered proliferative responses of B cells to various stimuli, abnormal vascular wound healing, and deficits in contextual and cued fear conditioning. ENU-induced mutations leadto impaired T cell-independent IgM responses. [provided by MGI curators]
Accession Number

NCBI RefSeq: NM_008855; MGI: 97596

MappedYes 
Amino Acid Change Methionine changed to Lysine
Institutional SourceBeutler Lab
Gene Model predicted gene model for protein(s): [ENSMUSP00000064812] [ENSMUSP00000070019] [ENSMUSP00000138788]
AlphaFold P68404
SMART Domains Protein: ENSMUSP00000064812
Gene: ENSMUSG00000052889
AA Change: M420K

DomainStartEndE-ValueType
low complexity region 4 16 N/A INTRINSIC
C1 37 86 7.11e-16 SMART
C1 102 151 1.42e-15 SMART
C2 172 275 1.05e-23 SMART
S_TKc 342 600 4.36e-97 SMART
S_TK_X 601 664 9.86e-27 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000064921)
SMART Domains Protein: ENSMUSP00000070019
Gene: ENSMUSG00000052889
AA Change: M420K

DomainStartEndE-ValueType
low complexity region 4 16 N/A INTRINSIC
C1 37 86 7.11e-16 SMART
C1 102 151 1.42e-15 SMART
C2 172 275 1.05e-23 SMART
S_TKc 342 600 4.36e-97 SMART
S_TK_X 601 663 6.27e-20 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000064989)
SMART Domains Protein: ENSMUSP00000138788
Gene: ENSMUSG00000052889
AA Change: M420K

DomainStartEndE-ValueType
low complexity region 4 16 N/A INTRINSIC
C1 37 86 7.11e-16 SMART
C1 102 151 1.42e-15 SMART
C2 172 275 1.05e-23 SMART
S_TKc 342 600 4.36e-97 SMART
S_TK_X 601 663 6.27e-20 SMART
Predicted Effect probably damaging

PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
(Using ENSMUST00000143692)
Meta Mutation Damage Score 0.9605 question?
Is this an essential gene? Non Essential (E-score: 0.000) question?
Phenotypic Category Autosomal Semidominant
Candidate Explorer Status loading ...
Single pedigree
Linkage Analysis Data
Penetrance  
Alleles Listed at MGI

All mutations/alleles(5) : Chemically induced (ENU)(2) Targeted(3)

Lab Alleles
AlleleSourceChrCoordTypePredicted EffectPPH Score
tilcara APN 7 122194228 missense probably damaging 1.00
IGL02045:Prkcb APN 7 122189390 missense probably damaging 1.00
IGL02273:Prkcb APN 7 122226990 missense probably damaging 1.00
IGL02638:Prkcb APN 7 122200063 splice site probably benign
IGL02962:Prkcb APN 7 122024270 splice site probably null
IGL03013:Prkcb APN 7 122226905 missense probably damaging 1.00
IGL03224:Prkcb APN 7 122116147 nonsense probably null
Baghdad UTSW 7 122226886 missense probably benign 0.07
Mesopotamia UTSW 7 121888737 missense probably damaging 1.00
Mosul UTSW 7 122116067 missense probably damaging 1.00
tigris UTSW 7 122024200 missense probably damaging 1.00
Tikrit UTSW 7 122226916 missense probably damaging 1.00
untied UTSW 7 122181662 missense possibly damaging 0.90
F5770:Prkcb UTSW 7 122127699 missense probably damaging 0.99
R0078:Prkcb UTSW 7 122189393 missense probably damaging 1.00
R0409:Prkcb UTSW 7 122024200 missense probably damaging 1.00
R0660:Prkcb UTSW 7 122024182 missense possibly damaging 0.56
R1462:Prkcb UTSW 7 122181672 missense probably damaging 1.00
R1462:Prkcb UTSW 7 122181672 missense probably damaging 1.00
R1480:Prkcb UTSW 7 122193865 missense probably damaging 1.00
R1518:Prkcb UTSW 7 122143854 critical splice acceptor site probably null
R1540:Prkcb UTSW 7 122226916 missense probably damaging 1.00
R1860:Prkcb UTSW 7 122167424 missense probably damaging 1.00
R3110:Prkcb UTSW 7 122116079 missense probably damaging 0.99
R3112:Prkcb UTSW 7 122116079 missense probably damaging 0.99
R4583:Prkcb UTSW 7 122056447 missense probably benign 0.32
R4847:Prkcb UTSW 7 122167372 missense probably benign 0.35
R5220:Prkcb UTSW 7 121888678 missense probably damaging 1.00
R5487:Prkcb UTSW 7 122199948 nonsense probably null
R5599:Prkcb UTSW 7 122181701 missense probably benign 0.17
R5946:Prkcb UTSW 7 122143926 missense probably benign
R6257:Prkcb UTSW 7 122167386 missense probably benign
R6590:Prkcb UTSW 7 121888737 missense probably damaging 1.00
R6618:Prkcb UTSW 7 122226886 missense probably benign 0.07
R6690:Prkcb UTSW 7 121888737 missense probably damaging 1.00
R6763:Prkcb UTSW 7 122193887 missense probably damaging 1.00
R7289:Prkcb UTSW 7 122143910 missense probably benign 0.04
R7414:Prkcb UTSW 7 122167450 missense possibly damaging 0.83
R7466:Prkcb UTSW 7 122116067 missense probably damaging 1.00
R7540:Prkcb UTSW 7 122167357 missense probably damaging 0.99
R8283:Prkcb UTSW 7 122199948 nonsense probably null
R9072:Prkcb UTSW 7 122127771 missense probably benign 0.14
R9483:Prkcb UTSW 7 122181663 missense probably damaging 0.99
R9670:Prkcb UTSW 7 122233070 nonsense probably null
V7581:Prkcb UTSW 7 122127699 missense probably damaging 0.99
X0061:Prkcb UTSW 7 122056529 missense probably benign 0.03
Z1177:Prkcb UTSW 7 122167419 missense possibly damaging 0.90
Mode of Inheritance Autosomal Semidominant
Local Stock
MMRRC Submission 038158-MU
Last Updated 2019-09-04 9:47 PM by Diantha La Vine
Record Created 2014-12-30 12:43 AM by Jin Huk Choi
Record Posted 2015-01-28
Phenotypic Description
Figure 1. Almonde mice exhibit diminished T-independent IgM responses to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll). IgM levels were determined by ELISA. Normalized data are shown. Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.

The Almonde phenotype was identified among N-Nitroso-N-ethylurea (ENU)-mutagenized G3 mice of the pedigree R1462, some of which showed a diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll) (Figure 1). 

Nature of Mutation
Figure 2. Linkage mapping of the reduced T-independent antibody response to NP-Ficoll using an additive model of inheritance. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 84 mutations (X-axis) identified in the G1 male of pedigree R1462.  Normalized phenotype data are shown for single locus linkage analysis with consideration of G2 dam identity.  Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.

Whole exome HiSeq sequencing of the G1 grandsire identified 84 mutations. The diminished T-independent antibody response to NP-Ficoll was linked by continuous variable mapping to a mutation in Prkcb: a T to A transversion at base pair 122,582,449 (v38) on chromosome 7, or base pair 293,370 in the GenBank genomic region NC_000073. Linkage was found with an additive model of linkage (P = 2.61 x 10-11), wherein two variant homozygotes and seven heterozygotes departed phenotypically from five homozygous reference mice (Figure 2). The mutation corresponds to residue 1,479 in the mRNA sequence NM_008855 within exon 11 of 17 total exons.

 
1463 CGCCTGTACTTTGTGATGGAGTATGTGAACGGG
415  -R--L--Y--F--V--M--E--Y--V--N--G-

The mutated nucleotide is indicated in red.  The mutation results in a methionine (M) to lysine (K) substitution at position 420 (M420K) in the PKCβ protein, and is strongly predicted by Polyphen-2 to cause loss of function (score = 1.000).

Illustration of Mutations in
Gene & Protein
Protein Prediction

Figure 3. Domain structure of conventional PKCs (amino acid numbering is for PKCβII). Key features of the regulatory domain include the pseudosubstrate motif (PS), the DAG-binding C1 domain, and the calcium-binding C2 domain. The kinase domain consists of the ATP-binding C3 domain, the substrate-binding domain and the V5 region. Key phosphorylation sites necessary for PKC activity are indicated in orange ovals. The residue altered by the Almonde mutation is shown in red. C, conserved region; V, variable region. Other mutations found in PRKCB are noted in red. Click on each mututation for more specific information.

PKCβ is a member of the protein kinase C (PKC) family of serine-threonine kinases. The PKC family belongs to the AGC-type kinase (protein kinase A/protein kinase G/protein kinase C) superfamily. PKC kinases share certain structural features including a highly conserved catalytic domain consisting of motifs required for ATP-substrate binding and catalysis, and a regulatory domain that maintains the enzyme in an inactive conformation. The regulatory and catalytic domains are attached to each other by a hinge region (Figure 3). Conventional PKCs (cPKCs) contain five variable (V) domains and four conserved (C) domains (1).

The Almonde mutation results in the substitution of a methionine for a lysine at amino acid 420 within the ATP-binding C3 domain within the kinase domain.

Please see the record Untied for information about Prkcb.

Putative Mechanism

In B cell receptor signaling, PKCβ functions to upregulate NF-κB activity and to promote B-cell activation, PKCβ can also directly inhibit Btk through a negative feedback loop (2). PKCβ specifically phosphorylates Btk at Ser180 within its Tec-homology (TH) region, leading to an inhibition of Btk membrane translocation and activation, and the downstream events that promote PKCβ activation. PKCβ activity also appears to play a role in mediating B cell activating factor (BAFF)-induced signals leading to B cell survival by phosphorylating the Akt kinase (also known as protein kinase B or PKB) and contributing to its activation. A targeted knockout of the Prkcb gene in mice resulted in animals with reduced numbers of mature peripheral B cells, a loss of peritoneal B-1 B cells, reduced T cell-independent antibody responses, as well as reduced function of various other immune cell types. The reduction of B cell antibody responses to TNP-Ficoll in Almonde mice suggests that the function of B-1 and/or MZ B cells is impaired in these animals with BCR signaling likely affected.  These phenotypes are consistent with the phenotypes observed in Prkcb-/-animals, which also exhibit reduced T cell-independent antibody responses along with severe impairment of B-1 cells (3).  The semidominant phenotype observed in Almonde mice may be due to the expression of nonfunctional, but appropriately localized, PKCβ proteins that are then able to inhibit the appropriate localization and function of wild type kinases.

Primers PCR Primer
Almonde_pcr_F: TACAGTGGCTCAGAGGTCACCCTTAG
Almonde_pcr_R: AGGTCTAGAATCTTGGCGGTTCCC

Sequencing Primer
Almonde_seq_F: GTCACCCTTAGTGGGTGC
Almonde_seq_R: tctctccctcaccgtacc
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 728 nucleotides is amplified (chromosome 7, + strand):


1   tacagtggct cagaggtcac ccttagtggg tgctgggccc tgcctgaaag aacactccgg
61  agttctggta gctctcatca taagtcccag ctactccctg ctccccgttt gggtcctgag
121 aaagtatggg gtgctgactt tggtgtttgc tctctttcct caggaccgcc tgtactttgt
181 gatggagtat gtgaacgggg gtgacctcat gtaccacatc caacaagttg gccgtttcaa
241 ggagccccat gctgtgtaag acagaccctc tttgtgccct ttccttggga taaatgattg
301 tcatttcctt taagttttag cctactaggg atttttttta ccccaacatc ccaggcttct
361 tctttgtgag cgttatttgg ccaaggacaa ctgtattctg ctcgctttga gatgtttctt
421 tatgttcctg aaggccagct aagaactcat agctctagac caatgactgt ttcataatct
481 ctaatttttt tttgcccctc catcctgaga aaggtcttat aacttccttg gggaatgtct
541 ttgcaggttt tttgtctgtt catttgctaa tatttattga gagcgttctt tgtgccagac
601 cctcgagtac tgaacaaaca ccatctcatt tcatcctgag cggtacggtg agggagagaa
661 tctcacccaa gctttacaat caagggcaac gagacccgga gggtgggaac cgccaagatt
721 ctagacct


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
AuthorsKuan-Wen Wang, Jin Huk Choi, Apiruck Watthanasurorot, Bruce Beutler