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REDK, a novel human regulatory erythroid kinase

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Presentation on theme: "REDK, a novel human regulatory erythroid kinase"— Presentation transcript:

1 REDK, a novel human regulatory erythroid kinase
by Kenneth A. Lord, Caretha L. Creasy, Andrew G. King, Caroline King, Brian M. Burns, John C. Lee, and Susan B. Dillon Blood Volume 95(9): May 1, 2000 ©2000 by American Society of Hematology

2 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

3 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

4 Tissue distribution of REDK message
Tissue distribution of REDK message.PolyA + RNA multiple-tissue Northern blots were hybridized to the REDK cDNA probe fragment. Tissue distribution of REDK message.PolyA + RNA multiple-tissue Northern blots were hybridized to the REDK cDNA probe fragment. Hybridizations to probes for β-actin and G3PDH are shown to illustrate loading consistency. H, heart; B, brain; Pl, placenta; Lu, lung; Lv, liver; Skm, skeletal muscle; K, kidney; Pn, pancreas; Sp, spleen; Th, thymus; Pr, prostate; Ts, testis; Ov, ovary; SI, small intestine; C, colon; PBL, peripheral blood leukocytes; LN, lymph node; BM, bone marrow; fLv, fetal liver. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

5 Expression of REDK message in bone marrow
Expression of REDK message in bone marrow.Total RNA from untreated (U) human bone marrow or from cells treated with stem cell factor (S), erythropoietin (E), thrombopoietin (T), granulocyte-colony stimulating factor (G), interleukin-3 (I3), or flt3 ligand (... Expression of REDK message in bone marrow.Total RNA from untreated (U) human bone marrow or from cells treated with stem cell factor (S), erythropoietin (E), thrombopoietin (T), granulocyte-colony stimulating factor (G), interleukin-3 (I3), or flt3 ligand (f3L) for 3 or 7 days was blotted and hybridized to the REDK cDNA probe. Hybridization to a DNA oligonucleotide probe for 28S RNA is shown to display relative loading of the RNA. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

6 Semiquantitative RT-PCR determination of REDK mRNA expression in different tissues.Total REDK PCR primer set was used to amplify a RED-specific product of 466 nucleotides after first-strand cDNA synthesis from pA + RNA. Semiquantitative RT-PCR determination of REDK mRNA expression in different tissues.Total REDK PCR primer set was used to amplify a RED-specific product of 466 nucleotides after first-strand cDNA synthesis from pA + RNA. Bands produced during exponential amplification were volume quantified and normalized to a G3PDH-specific product. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

7 Semiquantitative RT-PCR determination of the REDK-L and REDK-S messages.PCR amplification was performed on first-strand cDNA using sense primers specific for the long form or the short form, and a common antisense primer. Semiquantitative RT-PCR determination of the REDK-L and REDK-S messages.PCR amplification was performed on first-strand cDNA using sense primers specific for the long form or the short form, and a common antisense primer. Bands produced from exponential amplification were volume quantitated and normalized to G3PDH for comparison. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

8 Expression of REDK in CD34+ cells during erythroid differentiation
Expression of REDK in CD34+ cells during erythroid differentiation.Purified CD34+ cells were seeded in medium (20% FBS) containing 4 U/mL EPO and 50 ng/mL SCF. Cells were harvested for RNA extraction at the days indicated, and RT-PCR analysis was performed ... Expression of REDK in CD34+ cells during erythroid differentiation.Purified CD34+ cells were seeded in medium (20% FBS) containing 4 U/mL EPO and 50 ng/mL SCF. Cells were harvested for RNA extraction at the days indicated, and RT-PCR analysis was performed with REDK-L– and REDK-S–specific primers. Quantitation of the relative expression of the REDK-L and REDK-S messages was determined based on normalization to G3PDH. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

9 Detection of REDK protein by peptide antisera
Detection of REDK protein by peptide antisera.Whole-cell lysate from UT7-EPO cells was blotted with rabbit preimmune and immune sera after immunization with the Y3-52 peptide. Detection of REDK protein by peptide antisera.Whole-cell lysate from UT7-EPO cells was blotted with rabbit preimmune and immune sera after immunization with the Y3-52 peptide. The predicted molecular mass for REDK is 67 kd. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

10 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

11 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

12 REDK protein correlates with erythroid differentiation
REDK protein correlates with erythroid differentiation.Low-density mononuclear cells from a normal human donor were untreated (Unt) or treated for 7 days as shown. REDK protein correlates with erythroid differentiation.Low-density mononuclear cells from a normal human donor were untreated (Unt) or treated for 7 days as shown. Whole-cell protein lysates were analyzed for REDK protein by Western blotting. Comparable results were obtained from multiple donors. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

13 Recombinant REDK can phosphorylate myelin basic protein and histones
Recombinant REDK can phosphorylate myelin basic protein and histones.Nuclear protein extracts from log cells stably transfected with a tagged, recombinant REDK-S (clone S9) were immunoprecipitated by the T7-tag-specific sera. Recombinant REDK can phosphorylate myelin basic protein and histones.Nuclear protein extracts from log cells stably transfected with a tagged, recombinant REDK-S (clone S9) were immunoprecipitated by the T7-tag-specific sera. Immunoprecipitates were incubated with P-33-ATP and substrates to assess activity. Samples were collected in the linear range and analyzed as previously. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

14 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

15 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

16 Kenneth A. Lord et al. Blood 2000;95:2838-2846
©2000 by American Society of Hematology

17 The effect of antisense phosphorothioate DNA oligonucleotides to REDK is sequence-specific.Low-density bone marrow cells were incubated with REDK sense (S223), antisense (AS480), mutant antisense (AS480M1, AS480M2), or DYRK2 antisense (AS DYRK2) and were pl... The effect of antisense phosphorothioate DNA oligonucleotides to REDK is sequence-specific.Low-density bone marrow cells were incubated with REDK sense (S223), antisense (AS480), mutant antisense (AS480M1, AS480M2), or DYRK2 antisense (AS DYRK2) and were plated in methylcellulose-based medium with synergistic factors, as described. BFU-E (upper panel) and CFU-E (lower panel) were scored at 12 and 7 days, respectively. Kenneth A. Lord et al. Blood 2000;95: ©2000 by American Society of Hematology

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