Expression, purification, and biochemical characterization of Chk, a soluble protein tyrosine kinase.
CSK family contains two protein tyrosine kinases: Csk (C-terminal Src kinase) and Chk (Csk homologous kinase). They are responsible for phosphorylating Src family protein tyrosine kinases on a C-terminal Tyr (Tyr527) and negatively regulating their activities. However, Chk and Csk have different expression patterns, mechanisms of regulation, and different biological functions, and appear to play different roles in the development of breast cancer. To obtain pure human Chk for biochemical characterization, its coding region was amplified by polymerase chain reaction and expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The enzyme was highly expressed but unusually prone to proteolytic degradation during purification. Expression of the enzyme as a dual fusion protein with glutathione S-transferase on N-terminus and streptag, a 10 amino acid peptide, on C-terminus allowed purification of the full-length fusion protein. The purified enzyme was able to phosphorylate and inactivate Src. Chk (no inhibition up to 18.5 microM) and Csk (IC(50)= 1 microM) were differentially inhibited by PP2, probably due to the size difference of one residue (Thr265 in Csk versus Met304 in Chk) in the ATP-binding domain. The expression, purification, and initial characterizations of Chk provided an important step toward full characterization of Chk and Csk, two important enzymes in cellular regulation.
Ayrapetov
Marina K
MK
Department of Cell and Molecular Biology, University of Rhode Island, 117 Morrill Hall, 45 Lower College Road, Kingston, RI 02881, USA.
Lee
Sungsoo
S
Sun
Gongqin
G
D000595:Amino Acid Sequence; D015153:Blotting, Western; D000081247:CSK Tyrosine-Protein Kinase; D017931:DNA Primers / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D016392:Proto-Oncogene Proteins pp60(c-src); D011743:Pyrimidines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013917:Thrombin / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
C412373:AG 1879; D017931:DNA Primers; D004791:Enzyme Inhibitors; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; C085377:MATK protein, human; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; C000631346:CSK protein, human; D013917:Thrombin