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Characterization of a variant of PAC-1 in large granular lymphocyte leukemia
Ravi Kothapalli1, Sean J Yoder, Irina Kusmartseva
1Hematologic 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
Abstract:
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.
Insights
Researchers identified a novel, shorter variant of Phosphatase in activated T cells (PAC-1) overexpressed in large granular lymphocyte (LGL) leukemia. This variant, lacking intrinsic phosphatase activity, enhanced CL100 phosphatase activity in vitro, suggesting a potential role in LGL leukemia pathogenesis.
Area of Science:
- Molecular Biology
- Immunology
- Oncology
Background:
- Phosphatase in activated T cells (PAC-1) is a key regulator of MAP kinase activity.
- Constitutive PAC-1 expression is linked to HTLV-1 infection and T-cell malignancies.
- Large granular lymphocyte (LGL) leukemia is a T-cell malignancy characterized by abnormal lymphocyte proliferation.
Purpose of the Study:
- To investigate the role of PAC-1 related transcripts in LGL leukemia.
- To characterize a novel PAC-1 variant identified in LGL leukemia cells.
- To determine the enzymatic activity and potential function of the novel PAC-1 variant.
Main Methods:
- Screening of a LGL leukemia cDNA library using a PAC-1 probe.
- Molecular cloning and sequencing of a novel PAC-1 related transcript (clone 8).
- Expression and purification of the variant protein as a GST-fusion protein in E. coli.
- In vitro phosphatase assays to assess enzymatic activity and interaction with CL100.
Main Results:
- Identification and cloning of a novel PAC-1 related transcript (clone 8) with retained introns and excluded exons, resulting in a 170-amino acid protein.
- The purified GST-fusion protein (45 kDa) exhibited no intrinsic phosphatase activity.
- The purified variant protein enhanced the phosphatase activity of recombinant CL100 in vitro.
Conclusions:
- A novel, shorter PAC-1 variant is constitutively overexpressed in LGL leukemia.
- This variant lacks intrinsic phosphatase activity but can modulate the activity of other phosphatases.
- The precise physiological role and significance of this PAC-1 variant in LGL leukemia require further investigation.
