Platelet protein kinase C-theta deficiency with human RUNX1 mutation: PRKCQ is a transcriptional target of RUNX1.
OBJECTIVE
Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C- (PKC-, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC- in megakaryocytes and platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1.


RESULTS
In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region -1225 to -1056 bp containing a RUNX1 consensus site ACCGCA at -1088 to -1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC- protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC- protein were decreased by short interfering RNA knockdown of RUNX1.


CONCLUSIONS
Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC- deficiency associated with RUNX1 haplodeficiency.
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA.
Gauthami
G
Jalagadugula
Guangfen
G
Mao
Gurpreet
G
Kaur
Danny N
DN
Dhanasekaran
A Koneti
AK
Rao
D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D016384:Consensus Sequence; D050676:Core Binding Factor Alpha 2 Subunit / Q000097:blood / Q000235:genetics*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D008297:Male; D008533:Megakaryocytes / Q000201:enzymology*; D009154:Mutation; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000097:blood / Q000172:deficiency / Q000235:genetics*; D000074121:Protein Kinase C-theta; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism; D013921:Thrombocytopenia / Q000097:blood / Q000201:enzymology / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D055815:Young Adult
D050676:Core Binding Factor Alpha 2 Subunit; D007527:Isoenzymes; C493728:RUNX1 protein, human; D011994:Recombinant Proteins; C076233:PRKCQ protein, human; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta
J Biol Chem. 2007 Mar 9;282(10):7046-55
17210570
Blood. 1996 Jan 1;87(1):123-31
8547633
Nucleic Acids Res. 1983 Mar 11;11(5):1475-89
6828386
J Biol Chem. 1997 Nov 7;272(45):28704-11
9353339
Biochem Biophys Res Commun. 2009 Jul 17;385(1):22-7
19433059
Oncogene. 2006 Feb 2;25(5):781-94
16186797
J Biol Chem. 2010 Jul 23;285(30):23410-9
20479008
Blood. 2004 Dec 1;104(12):3565-72
15297309
Blood. 2009 Mar 12;113(11):2557-67
19164598
Blood. 2001 Jan 1;97(1):154-61
11133755
J Cell Physiol. 2006 Jun;207(3):582-93
16250015
Blood. 2009 Apr 9;113(15):3577-84
19190246
J Clin Invest. 1990 Apr;85(4):1072-84
2318965
J Biol Chem. 2005 Jan 7;280(1):644-53
15536078
Blood. 2002 May 15;99(10):3579-84
11986211
Nature. 1988 Jun 2;333(6172):470-3
2897630
Blood. 2010 Apr 15;115(15):3128-35
20181616
J Clin Invest. 2009 Feb;119(2):399-407
19147982
Arterioscler Thromb Vasc Biol. 2009 May;29(5):699-705
19213940
J Thromb Haemost. 2007 Jan;5(1):146-54
17059412
Arterioscler Thromb Vasc Biol. 2000 Jun;20(6):1651-60
10845885
Nature. 1993 May 27;363(6427):309-10
8497315
FEBS Lett. 1982 Nov 1;148(1):21-6
6816632
Br J Haematol. 1996 Jun;93(4):762-71
8703801
Exp Hematol. 1996 Nov;24(13):1501-8
8950233
Blood. 2002 Feb 15;99(4):1364-72
11830488
J Biol Chem. 2003 Jul 18;278(29):26374-9
12724315
PLoS One. 2008;3(11):e3793
19030108
Blood. 2010 Dec 23;116(26):6037-45
20876458
Thromb Haemost. 2002 Jan;87(1):129-33
11848441
Biochem J. 1991 Jan 1;273(Pt 1):115-20
1846526
Br J Haematol. 1996 Mar;92(3):530-6
8616013
Blood. 2006 Dec 15;108(13):4035-44
16940418
Exp Hematol. 2004 Oct;32(10):991-9
15504554
J Thromb Haemost. 2006 Mar;4(3):648-55
16460447
Blood. 1996 Feb 15;87(4):1368-76
8608226
Biochem Biophys Res Commun. 1999 Oct 5;263(3):603-9
10512725
J Thromb Haemost. 2011 Feb;9(2):383-91
21129147
J Biol Chem. 2004 Jan 9;279(2):825-30
14561740
J Thromb Haemost. 2010 Mar;8(3):454-62
20002545
Nat Med. 2004 Mar;10(3):299-304
14966519
Oncogene. 2004 May 24;23(24):4238-48
15156179
Blood. 2004 Feb 1;103(3):948-54
14525764
J Biol Chem. 2001 Oct 19;276(42):39379-85
11495897
Biochem J. 1993 Jan 1;289 ( Pt 1):277-82
8424766
Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3444-9
8622955
PLoS One. 2008 Sep 25;3(9):e3277
18815612
Nat Genet. 1999 Oct;23(2):166-75
10508512