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Protein

Protein kinase C beta type

Gene

Prkcb

Organism
Rattus norvegicus (Rat)
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase involved in various cellular processes such as regulation of the B-cell receptor (BCR) signalosome, oxidative stress-induced apoptosis, androgen receptor-dependent transcription regulation, insulin signaling and endothelial cells proliferation. Plays a key role in B-cell activation by regulating BCR-induced NF-kappa-B activation. Mediates the activation of the canonical NF-kappa-B pathway (NFKB1) by direct phosphorylation of CARD11/CARMA1 at 'Ser-559', 'Ser-644' and 'Ser-652'. Phosphorylation induces CARD11/CARMA1 association with lipid rafts and recruitment of the BCL10-MALT1 complex as well as MAP3K7/TAK1, which then activates IKK complex, resulting in nuclear translocation and activation of NFKB1. Plays a direct role in the negative feedback regulation of the BCR signaling, by down-modulating BTK function via direct phosphorylation of BTK at 'Ser-180', which results in the alteration of BTK plasma membrane localization and in turn inhibition of BTK activity. Involved in apoptosis following oxidative damage: in case of oxidative conditions, specifically phosphorylates 'Ser-36' of isoform p66Shc of SHC1, leading to mitochondrial accumulation of p66Shc, where p66Shc acts as a reactive oxygen species producer. Acts as a coactivator of androgen receptor (ANDR)-dependent transcription, by being recruited to ANDR target genes and specifically mediating phosphorylation of 'Thr-6' of histone H3 (H3T6ph), a specific tag for epigenetic transcriptional activation that prevents demethylation of histone H3 'Lys-4' (H3K4me) by LSD1/KDM1A. In insulin signaling, may function downstream of IRS1 in muscle cells and mediate insulin-dependent DNA synthesis through the RAF1-MAPK/ERK signaling cascade. May participate in the regulation of glucose transport in adipocytes by negatively modulating the insulin-stimulated translocation of the glucose transporter SLC2A4/GLUT4. Under high glucose in pancreatic beta-cells, is probably involved in the inhibition of the insulin gene transcription, via regulation of MYC expression. In endothelial cells, activation of PRKCB induces increased phosphorylation of RB1, increased VEGFA-induced cell proliferation, and inhibits PI3K/AKT-dependent nitric oxide synthase (NOS3/eNOS) regulation by insulin, which causes endothelial dysfunction. Also involved in triglyceride homeostasis (By similarity). Phosphorylates ATF2 which promotes cooperation between ATF2 and JUN, activating transcription.By similarity3 Publications

Catalytic activityi

ATP + a protein = ADP + a phosphoprotein.

Cofactori

Ca2+2 PublicationsNote: Binds 3 Ca2+ ions per subunit. The ions are bound to the C2 domain.2 Publications

Enzyme regulationi

Classical (or conventional) PKCs (PRKCA, PRKCB and PRKCG) are activated by calcium and diacylglycerol (DAG) in the presence of phosphatidylserine. Three specific sites; Thr-500 (activation loop of the kinase domain), Thr-642 (turn motif) and Ser-661 (hydrophobic region), need to be phosphorylated for its full activation. Specifically inhibited by enzastaurin (LY317615) (By similarity).By similarity

Sites

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Metal bindingi186Calcium 1; via carbonyl oxygen2 Publications1
Metal bindingi187Calcium 12 Publications1
Metal bindingi187Calcium 22 Publications1
Metal bindingi193Calcium 22 Publications1
Metal bindingi246Calcium 12 Publications1
Metal bindingi246Calcium 22 Publications1
Metal bindingi247Calcium 2; via carbonyl oxygen2 Publications1
Metal bindingi248Calcium 12 Publications1
Metal bindingi248Calcium 22 Publications1
Metal bindingi248Calcium 32 Publications1
Metal bindingi251Calcium 32 Publications1
Metal bindingi252Calcium 3; via carbonyl oxygen2 Publications1
Metal bindingi254Calcium 12 Publications1
Metal bindingi254Calcium 32 Publications1
Binding sitei371ATPPROSITE-ProRule annotation1
Active sitei466Proton acceptorPROSITE-ProRule annotation1

Regions

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Zinc fingeri36 – 86Phorbol-ester/DAG-type 1PROSITE-ProRule annotationAdd BLAST51
Zinc fingeri101 – 151Phorbol-ester/DAG-type 2PROSITE-ProRule annotationAdd BLAST51
Nucleotide bindingi348 – 356ATPPROSITE-ProRule annotation9

GO - Molecular functioni

GO - Biological processi

Complete GO annotation...

Keywords - Molecular functioni

Chromatin regulator, Kinase, Serine/threonine-protein kinase, Transferase

Keywords - Biological processi

Adaptive immunity, Apoptosis, Immunity, Transcription, Transcription regulation

Keywords - Ligandi

ATP-binding, Calcium, Metal-binding, Nucleotide-binding, Zinc

Names & Taxonomyi

Protein namesi
Recommended name:
Protein kinase C beta type (EC:2.7.11.13)
Short name:
PKC-B
Short name:
PKC-beta
Gene namesi
Name:Prkcb
Synonyms:Pkcb, Prkcb1
OrganismiRattus norvegicus (Rat)
Taxonomic identifieri10116 [NCBI]
Taxonomic lineageiEukaryotaMetazoaChordataCraniataVertebrataEuteleostomiMammaliaEutheriaEuarchontogliresGliresRodentiaSciurognathiMuroideaMuridaeMurinaeRattus
Proteomesi
  • UP000002494 Componenti: Unplaced

Organism-specific databases

RGDi3396. Prkcb.

Subcellular locationi

GO - Cellular componenti

  • brush border membrane Source: RGD
  • centrosome Source: RGD
  • cytosol Source: RGD
  • nucleus Source: UniProtKB
  • plasma membrane Source: RGD
Complete GO annotation...

Keywords - Cellular componenti

Cytoplasm, Membrane, Nucleus

Pathology & Biotechi

Mutagenesis

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Mutagenesisi16 – 17ST → AA: No effect. 1 Publication2
Mutagenesisi314T → A: No effect; when associated with A-323. 1 Publication1
Mutagenesisi324T → A: No effect; when associated with A-313. 1 Publication1
Mutagenesisi500T → E: 50% increase of enzymatic activity. 1 Publication1
Mutagenesisi500T → S: 50% decrease of enzymatic activity. 1 Publication1
Mutagenesisi500T → V or D: Loss of enzymatic activity. 1 Publication1
Mutagenesisi635T → A: Loss of enzymatic activity; when associated with T-643 change in subcellular location, loss of PMA-induced down-regulation and loss of enzymatic activity. 2 Publications1
Mutagenesisi642T → A: Loss of enzymatic activity; when associated with T-636 change in subcellular location, loss of PMA-induced down-regulation and loss of enzymatic activity. 2 Publications1

Chemistry databases

ChEMBLiCHEMBL3020.

PTM / Processingi

Molecule processing

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Initiator methionineiRemovedBy similarity
ChainiPRO_00000556872 – 671Protein kinase C beta typeAdd BLAST670

Amino acid modifications

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Modified residuei2N-acetylalanineBy similarity1
Modified residuei11PhosphoserineCombined sources1
Modified residuei16Phosphoserine; by autocatalysis1 Publication1
Modified residuei17Phosphothreonine; by autocatalysis1 Publication1
Modified residuei206PhosphoserineBy similarity1
Modified residuei250Phosphothreonine; by autocatalysisBy similarity1
Modified residuei314Phosphothreonine; by autocatalysis1 Publication1
Modified residuei324Phosphothreonine; by autocatalysis1 Publication1
Modified residuei500Phosphothreonine; by PDPK13 Publications1
Modified residuei504PhosphothreonineBy similarity1
Modified residuei635Phosphothreonine; by autocatalysis1 Publication1
Modified residuei642Phosphothreonine; by autocatalysisCombined sources3 Publications1
Modified residuei661Phosphoserine; by autocatalysis2 Publications1
Modified residuei662Phosphotyrosine; by SYKBy similarity1
Isoform Beta-II (identifier: P68403-2)
Modified residuei641PhosphothreonineCombined sources1
Modified residuei654PhosphoserineCombined sources1
Modified residuei660PhosphoserineCombined sources1
Modified residuei664PhosphoserineCombined sources1

Post-translational modificationi

Phosphorylation on Thr-500 of isoform beta-I, within the activation loop, renders it competent to autophosphorylate. Subsequent autophosphorylation of Thr-642 maintains catalytic competence, and autophosphorylation on Ser-661 appears to release the kinase into the cytosol. Similarly, isoform beta-II is autophosphorylated on 'Thr-641' and 'Ser-660', subsequent to phosphorylation on Thr-500. Autophosphorylated on other sites i.e. in the N-terminal and hinge regions have no effect on enzyme activity. Phosphorylation at Tyr-662 by SYK induces binding with GRB2 and contributes to the activation of MAPK/ERK signaling cascade.4 Publications

Keywords - PTMi

Acetylation, Phosphoprotein

Proteomic databases

PaxDbiP68403.
PRIDEiP68403.

PTM databases

iPTMnetiP68403.

Expressioni

Gene expression databases

BgeeiENSRNOG00000012061.

Interactioni

Subunit structurei

Interacts with PDK1. Interacts in vitro with PRKCBP1. Interacts with PHLPP1 and PHLPP2; both proteins mediate its dephosphorylation. Interacts with KDM1A/LSD1, PKN1 and ANDR (By similarity).By similarity

Binary interactionsi

WithEntry#Exp.IntActNotes
PtpraP180523EBI-397072,EBI-6597520From a different organism.
TRIM41Q8WV44-23EBI-397092,EBI-726015From a different organism.

GO - Molecular functioni

Protein-protein interaction databases

BioGridi247103. 11 interactors.
IntActiP68403. 4 interactors.
MINTiMINT-4099933.
STRINGi10116.ENSRNOP00000016417.

Chemistry databases

BindingDBiP68403.

Structurei

Secondary structure

1671
Legend: HelixTurnBeta strandPDB Structure known for this area
Show more details
Feature keyPosition(s)DescriptionActionsGraphical viewLength
Beta strandi161 – 182Combined sources22
Beta strandi194 – 202Combined sources9
Beta strandi222 – 230Combined sources9
Helixi234 – 237Combined sources4
Beta strandi239 – 246Combined sources8
Beta strandi249 – 251Combined sources3
Beta strandi254 – 262Combined sources9
Helixi263 – 266Combined sources4
Beta strandi271 – 276Combined sources6
Helixi280 – 283Combined sources4

3D structure databases

Select the link destinations:
PDBei
RCSB PDBi
PDBji
Links Updated
PDB entryMethodResolution (Å)ChainPositionsPDBsum
1A25X-ray2.70A/B154-289[»]
3PFQX-ray4.00A1-661[»]
ProteinModelPortaliP68403.
SMRiP68403.
ModBaseiSearch...
MobiDBiSearch...

Miscellaneous databases

EvolutionaryTraceiP68403.

Family & Domainsi

Domains and Repeats

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Domaini173 – 260C2PROSITE-ProRule annotationAdd BLAST88
Domaini342 – 600Protein kinasePROSITE-ProRule annotationAdd BLAST259
Domaini601 – 671AGC-kinase C-terminalAdd BLAST71

Sequence similaritiesi

Contains 1 AGC-kinase C-terminal domain.Curated
Contains 1 C2 domain.PROSITE-ProRule annotation
Contains 2 phorbol-ester/DAG-type zinc fingers.PROSITE-ProRule annotation
Contains 1 protein kinase domain.PROSITE-ProRule annotation

Zinc finger

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Zinc fingeri36 – 86Phorbol-ester/DAG-type 1PROSITE-ProRule annotationAdd BLAST51
Zinc fingeri101 – 151Phorbol-ester/DAG-type 2PROSITE-ProRule annotationAdd BLAST51

Keywords - Domaini

Repeat, Zinc-finger

Phylogenomic databases

eggNOGiKOG0694. Eukaryota.
ENOG410XNPH. LUCA.
HOGENOMiHOG000233022.
HOVERGENiHBG108317.
InParanoidiP68403.
KOiK19662.
PhylomeDBiP68403.

Family and domain databases

CDDicd00029. C1. 2 hits.
Gene3Di2.60.40.150. 1 hit.
InterProiIPR000961. AGC-kinase_C.
IPR000008. C2_dom.
IPR020454. DAG/PE-bd.
IPR011009. Kinase-like_dom.
IPR002219. PE/DAG-bd.
IPR017892. Pkinase_C.
IPR000719. Prot_kinase_dom.
IPR017441. Protein_kinase_ATP_BS.
IPR014375. Protein_kinase_C_a/b/g.
IPR008271. Ser/Thr_kinase_AS.
[Graphical view]
PfamiPF00130. C1_1. 2 hits.
PF00168. C2. 1 hit.
PF00069. Pkinase. 1 hit.
PF00433. Pkinase_C. 1 hit.
[Graphical view]
PIRSFiPIRSF000550. PKC_alpha. 1 hit.
PRINTSiPR00360. C2DOMAIN.
PR00008. DAGPEDOMAIN.
SMARTiSM00109. C1. 2 hits.
SM00239. C2. 1 hit.
SM00133. S_TK_X. 1 hit.
SM00220. S_TKc. 1 hit.
[Graphical view]
SUPFAMiSSF49562. SSF49562. 1 hit.
SSF56112. SSF56112. 1 hit.
PROSITEiPS51285. AGC_KINASE_CTER. 1 hit.
PS50004. C2. 1 hit.
PS00107. PROTEIN_KINASE_ATP. 1 hit.
PS50011. PROTEIN_KINASE_DOM. 1 hit.
PS00108. PROTEIN_KINASE_ST. 1 hit.
PS00479. ZF_DAG_PE_1. 2 hits.
PS50081. ZF_DAG_PE_2. 2 hits.
[Graphical view]

Sequences (2)i

Sequence statusi: Complete.

Sequence processingi: The displayed sequence is further processed into a mature form.

This entry describes 2 isoformsi produced by alternative splicing. AlignAdd to basket

Isoform Beta-I (identifier: P68403-1) [UniParc]FASTAAdd to basket

This isoform has been chosen as the 'canonical' sequence. All positional information in this entry refers to it. This is also the sequence that appears in the downloadable versions of the entry.

« Hide

        10         20         30         40         50
MADPAAGPPP SEGEESTVRF ARKGALRQKN VHEVKNHKFT ARFFKQPTFC
60 70 80 90 100
SHCTDFIWGF GKQGFQCQVC CFVVHKRCHE FVTFSCPGAD KGPASDDPRS
110 120 130 140 150
KHKFKIHTYS SPTFCDHCGS LLYGLIHQGM KCDTCMMNVH KRCVMNVPSL
160 170 180 190 200
CGTDHTERRG RIYIQAHIDR EVLIVVVRDA KNLVPMDPNG LSDPYVKLKL
210 220 230 240 250
IPDPKSESKQ KTKTIKCSLN PEWNETFRFQ LKESDKDRRL SVEIWDWDLT
260 270 280 290 300
SRNDFMGSLS FGISELQKAG VDGWFKLLSQ EEGEYFNVPV PPEGSEGNEE
310 320 330 340 350
LRQKFERAKI GQGTKAPEEK TANTISKFDN NGNRDRMKLT DFNFLMVLGK
360 370 380 390 400
GSFGKVMLSE RKGTDELYAV KILKKDVVIQ DDDVECTMVE KRVLALPGKP
410 420 430 440 450
PFLTQLHSCF QTMDRLYFVM EYVNGGDLMY HIQQVGRFKE PHAVFYAAEI
460 470 480 490 500
AIGLFFLQSK GIIYRDLKLD NVMLDSEGHI KIADFGMCKE NIWDGVTTKT
510 520 530 540 550
FCGTPDYIAP EIIAYQPYGK SVDWWAFGVL LYEMLAGQAP FEGEDEDELF
560 570 580 590 600
QSIMEHNVAY PKSMSKEAVA ICKGLMTKHP GKRLGCGPEG ERDIKEHAFF
610 620 630 640 650
RYIDWEKLER KEIQPPYKPK ARDKRDTSNF DKEFTRQPVE LTPTDKLFIM
660 670
NLDQNEFAGF SYTNPEFVIN V
Length:671
Mass (Da):76,751
Last modified:January 23, 2007 - v3
Checksum:iA1935030F758513C
GO
Isoform Beta-II (identifier: P68403-2) [UniParc] [UniParc]FASTAAdd to basket

The sequence of this isoform differs from the canonical sequence as follows:
     622-671: RDKRDTSNFD...YTNPEFVINV → CGRNAENFDR...SEFLKPEVKS

Show »
Length:673
Mass (Da):76,894
Checksum:iA65573AAF6E224C7
GO

Experimental Info

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Sequence conflicti25A → P in AAA41868 (PubMed:3345563).Curated1
Sequence conflicti126I → V in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti138N → S in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti141K → R in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti149S → G in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti164I → V in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti170 – 171RE → GG in AAA41875 (PubMed:3755379).Curated2
Sequence conflicti174I → V in AAA41875 (PubMed:3755379).Curated1
Sequence conflicti294G → E in AAA41868 (PubMed:3345563).Curated1
Sequence conflicti419V → M in CAA28035 (PubMed:2428667).Curated1
Sequence conflicti419V → M in CAA28036 (PubMed:2428667).Curated1
Sequence conflicti628Missing in AAA41865 (PubMed:3755379).Curated1
Sequence conflicti640Missing in AAA41865 (PubMed:3755379).Curated1

Alternative sequence

Feature keyPosition(s)DescriptionActionsGraphical viewLength
Alternative sequenceiVSP_004739622 – 671RDKRD…FVINV → CGRNAENFDRFFTRHPPVLT PPDQEVIRNIDQSEFEGFSF VNSEFLKPEVKS in isoform Beta-II. 2 PublicationsAdd BLAST50

Sequence databases

Select the link destinations:
EMBLi
GenBanki
DDBJi
Links Updated
M13706 mRNA. Translation: AAA41875.1.
K03485 mRNA. Translation: AAA41864.1.
K03486 mRNA. Translation: AAA41865.1.
X04439 Genomic DNA. Translation: CAA28035.1.
X04440 mRNA. Translation: CAA28036.1.
M19007 mRNA. Translation: AAA41868.1.
X04139 mRNA. Translation: CAA27756.1.
M15522 mRNA. Translation: AAA41876.1.
PIRiA00622. KIRTC1.
RefSeqiNP_001165776.1. NM_001172305.1.
NP_036845.3. NM_012713.3.
UniGeneiRn.91118.

Genome annotation databases

GeneIDi25023.
KEGGirno:25023.
UCSCiRGD:3396. rat.

Keywords - Coding sequence diversityi

Alternative splicing

Cross-referencesi

Sequence databases

Select the link destinations:
EMBLi
GenBanki
DDBJi
Links Updated
M13706 mRNA. Translation: AAA41875.1.
K03485 mRNA. Translation: AAA41864.1.
K03486 mRNA. Translation: AAA41865.1.
X04439 Genomic DNA. Translation: CAA28035.1.
X04440 mRNA. Translation: CAA28036.1.
M19007 mRNA. Translation: AAA41868.1.
X04139 mRNA. Translation: CAA27756.1.
M15522 mRNA. Translation: AAA41876.1.
PIRiA00622. KIRTC1.
RefSeqiNP_001165776.1. NM_001172305.1.
NP_036845.3. NM_012713.3.
UniGeneiRn.91118.

3D structure databases

Select the link destinations:
PDBei
RCSB PDBi
PDBji
Links Updated
PDB entryMethodResolution (Å)ChainPositionsPDBsum
1A25X-ray2.70A/B154-289[»]
3PFQX-ray4.00A1-661[»]
ProteinModelPortaliP68403.
SMRiP68403.
ModBaseiSearch...
MobiDBiSearch...

Protein-protein interaction databases

BioGridi247103. 11 interactors.
IntActiP68403. 4 interactors.
MINTiMINT-4099933.
STRINGi10116.ENSRNOP00000016417.

Chemistry databases

BindingDBiP68403.
ChEMBLiCHEMBL3020.

PTM databases

iPTMnetiP68403.

Proteomic databases

PaxDbiP68403.
PRIDEiP68403.

Protocols and materials databases

Structural Biology KnowledgebaseSearch...

Genome annotation databases

GeneIDi25023.
KEGGirno:25023.
UCSCiRGD:3396. rat.

Organism-specific databases

CTDi5579.
RGDi3396. Prkcb.

Phylogenomic databases

eggNOGiKOG0694. Eukaryota.
ENOG410XNPH. LUCA.
HOGENOMiHOG000233022.
HOVERGENiHBG108317.
InParanoidiP68403.
KOiK19662.
PhylomeDBiP68403.

Miscellaneous databases

EvolutionaryTraceiP68403.
PROiP68403.

Gene expression databases

BgeeiENSRNOG00000012061.

Family and domain databases

CDDicd00029. C1. 2 hits.
Gene3Di2.60.40.150. 1 hit.
InterProiIPR000961. AGC-kinase_C.
IPR000008. C2_dom.
IPR020454. DAG/PE-bd.
IPR011009. Kinase-like_dom.
IPR002219. PE/DAG-bd.
IPR017892. Pkinase_C.
IPR000719. Prot_kinase_dom.
IPR017441. Protein_kinase_ATP_BS.
IPR014375. Protein_kinase_C_a/b/g.
IPR008271. Ser/Thr_kinase_AS.
[Graphical view]
PfamiPF00130. C1_1. 2 hits.
PF00168. C2. 1 hit.
PF00069. Pkinase. 1 hit.
PF00433. Pkinase_C. 1 hit.
[Graphical view]
PIRSFiPIRSF000550. PKC_alpha. 1 hit.
PRINTSiPR00360. C2DOMAIN.
PR00008. DAGPEDOMAIN.
SMARTiSM00109. C1. 2 hits.
SM00239. C2. 1 hit.
SM00133. S_TK_X. 1 hit.
SM00220. S_TKc. 1 hit.
[Graphical view]
SUPFAMiSSF49562. SSF49562. 1 hit.
SSF56112. SSF56112. 1 hit.
PROSITEiPS51285. AGC_KINASE_CTER. 1 hit.
PS50004. C2. 1 hit.
PS00107. PROTEIN_KINASE_ATP. 1 hit.
PS50011. PROTEIN_KINASE_DOM. 1 hit.
PS00108. PROTEIN_KINASE_ST. 1 hit.
PS00479. ZF_DAG_PE_1. 2 hits.
PS50081. ZF_DAG_PE_2. 2 hits.
[Graphical view]
ProtoNetiSearch...

Entry informationi

Entry nameiKPCB_RAT
AccessioniPrimary (citable) accession number: P68403
Secondary accession number(s): P04410, P04411
Entry historyi
Integrated into UniProtKB/Swiss-Prot: August 13, 1987
Last sequence update: January 23, 2007
Last modified: November 30, 2016
This is version 133 of the entry and version 3 of the sequence. [Complete history]
Entry statusiReviewed (UniProtKB/Swiss-Prot)
Annotation programChordata Protein Annotation Program

Miscellaneousi

Keywords - Technical termi

3D-structure, Complete proteome, Direct protein sequencing, Reference proteome

Documents

  1. PDB cross-references
    Index of Protein Data Bank (PDB) cross-references
  2. SIMILARITY comments
    Index of protein domains and families

Similar proteinsi

Links to similar proteins from the UniProt Reference Clusters (UniRef) at 100%, 90% and 50% sequence identity:
100%UniRef100 combines identical sequences and sub-fragments with 11 or more residues from any organism into one UniRef entry.
90%UniRef90 is built by clustering UniRef100 sequences that have at least 90% sequence identity to, and 80% overlap with, the longest sequence (a.k.a seed sequence).
50%UniRef50 is built by clustering UniRef90 seed sequences that have at least 50% sequence identity to, and 80% overlap with, the longest sequence in the cluster.