[Screening ubiquitin/ribosomal protein S27a by yeast two-hybridization system].
OBJECTIVE
Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession. Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth. In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library.


METHODS
The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR). Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I. The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments. Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcript method was used to construct a cDNA library in yeast cells. Library plasmid was named AD plasmid. BD plasmid and AD plasmid were co-transformed into competent yeast AH109. Yeast two-hybridization was used to screen positive clones.


RESULTS
Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened. DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%).


CONCLUSIONS
Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells.
Huang
Gong-Hua
GH
Institute of Biochemistry and Molecular Biology, Guangdong Medical College, Zhanjiang, Guangdong 524-023, P.R. China.
Liang
Jing-Yao
JY
Chen
Bi
B
Liu
Xin-Guang
XG
Liang
Nian-Ci
NC
D001483:Base Sequence; D047390:Casein Kinase II / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D025801:Ubiquitin; C096227:ribosomal protein S27a; D047390:Casein Kinase II