Characterization of a variant of PAC-1 in large granular lymphocyte leukemia.
Phosphatase in activated T cells (PAC-1) is a mitogen-induced early responsive gene. It encodes a 32 kDa tyrosine-threonine dual specificity phosphatase. Constitutive expression of PAC-1 leads to an inhibition of MAP kinase activity in vivo. Such constitutive expression was reported in HTLV-1 infected cell lines. In the present study, we observed the constitutive over-expression of two transcripts related to PAC-1 in large granular lymphocyte (LGL) leukemia. By screening a LGL leukemia cDNA library using the 3' end of a PAC-1 probe, we obtained a clone (clone 8) which retains one and one half introns, excludes two exons, and matches one hundred percent with a DNA sequence on chromosome 2. The deduced amino acid sequence of the predicted protein contains 170 amino acids and is 144 amino acids shorter than PAC-1. When we expressed this protein in Escherichia coli as a GST-fusion protein, a 45 kDa (19 kDa PAC-1 variant+26 kDa GST protein) protein was obtained. The expressed protein was purified to near homogeneity by using a glutathione affinity column. The purified protein did not have any intrinsic phosphatase activity when assayed in vitro. But when this purified protein was added to a phosphatase assay system in combination with a recombinant dual specificity phosphatase, CL100, enhanced phosphatase activity was observed. The significance of the constitutive over-expression and its physiological role of this protein remain to be established in leukemic LGL.
Kothapalli
Ravi
R
Hematologic Malignancies Program, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, MRC Room No. 2067 B, 12902 Magnolia Drive, Tampa, FL 33612, USA. kothapar@moffitt.usf.edu
Yoder
Sean J
SJ
Kusmartseva
Irina
I
Loughran
Thomas P
TP
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D054638:Dual Specificity Phosphatase 1; D054641:Dual Specificity Phosphatase 2; D020224:Expressed Sequence Tags; D015723:Gene Library; D014644:Genetic Variation / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017874:Immediate-Early Proteins / Q000378:metabolism; D007438:Introns / Q000235:genetics; D007945:Leukemia, Lymphoid / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D017027:Protein Tyrosine Phosphatases / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
D018797:Cell Cycle Proteins; D017874:Immediate-Early Proteins; D012333:RNA, Messenger; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; C517019:DUSP1 protein, human; C517032:DUSP2 protein, human; D054638:Dual Specificity Phosphatase 1; D054641:Dual Specificity Phosphatase 2; D017027:Protein Tyrosine Phosphatases