ABSTRACT
Chronic myelogenous leukemia (CML) is a malignancy of the human hematopoietic stem cell (HSC) caused by the p210BCR/ABL oncoprotein. Although alternative splicing of pre-mRNA is a critical determinant of a cell's protein repertoire, it has not been associated with CML pathogenesis. We identified a BCR/ABL-dependent increase in expression of multiple genes involved in pre-mRNA splicing (eg SRPK1, RNA Helicase II/Gu, and hnRNPA2/B1) by subtractive hybridization of cDNA from p210BCR/ABL-eGFP vs eGFP-transduced umbilical cord blood CD34+ cells. beta1-integrin signaling is important to HSC maintenance and proliferation/differentiation, and is abnormal in CML. As an example of how changes in pre-mRNA processing might contribute to CML pathogenesis, we observed alternative splicing of a gene for a beta1-integrin-responsive nonreceptor tyrosine kinase (PYK2), resulting in increased expression of full-length Pyk2 in BCR/ABL-containing cells. Treatment of p210BCR/ABL-positive cells with the Abl-specific tyrosine kinase inhibitor STI571 reverted PYK2 splicing to a configuration more consistent with normal cells, and correlated with decreased expression of BCR/ABL-induced proteins involved in pre-mRNA processing. Whether altered PYK2 splicing contributes to CML pathogenesis remains undetermined; however, we propose that generic changes in pre-mRNA splicing as a result of p210BCR/ABL kinase activity may contribute to CML pathogenesis.
Subject(s)
Fusion Proteins, bcr-abl/physiology , Hematopoietic Stem Cells/pathology , Protein-Tyrosine Kinases/genetics , RNA Splicing , Antigens, CD34 , Focal Adhesion Kinase 2 , Hematopoietic Stem Cells/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/etiology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Leukemia, Myeloid, Chronic-Phase/etiology , Leukemia, Myeloid, Chronic-Phase/genetics , Leukemia, Myeloid, Chronic-Phase/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , RNA Precursors/geneticsABSTRACT
Insulin regulates the activity of both protein kinases and phosphatases. Little is known concerning the subcellular effects of insulin on phosphatase activity and how it is affected by insulin resistance. The purpose of this study was to determine insulin-stimulated subcellular changes in phosphatase activity and how they are affected by insulin resistance. We used an in vitro fatty acid (palmitate) induced insulin resistance model, differential centrifugation to fractionate rat adipocytes, and a malachite green phosphatase assay using peptide substrates to measure enzyme activity. Overall, insulin alone had no effect on adipocyte tyrosine phosphatase activity; however, subcellularly, insulin increased plasma membrane adipocyte tyrosine phosphatase activity 78 +/- 26% (n = 4, P < 0.007), and decreased high-density microsome adipocyte tyrosine phosphatase activity 42 +/- 13% (n = 4, P < 0.005). Although insulin resistance induced specific changes in basal tyrosine phosphatase activity, insulin-stimulated changes were not significantly altered by insulin resistance. Insulin-stimulated overall serine/threonine phosphatase activity by 16 +/- 5% (n = 4, P < 0.005), which was blocked in insulin resistance. Subcellularly, insulin increased plasma membrane and crude nuclear fraction serine/threonine phosphatase activities by 59 +/- 19% (n = 4, P < 0. 005) and 21 +/- 7% (n = 4, P < 0.007), respectively. This increase in plasma membrane fractions was inhibited 23 +/- 7% (n = 4, P < 0. 05) by palmitate. Furthermore, insulin increased cytosolic protein phosphatase-1 (PP-1) activity 160 +/- 50% (n = 3, P < 0.015), and palmitate did not significantly reduce this activity. However, palmitate did reduce insulin-treated low-density microsome protein phosphatase-1 activity by 28 +/- 6% (n = 3, P < 0.04). Insulin completely inhibited protein phosphatase-2A activity in the cytosol and increased crude nuclear fraction protein phosphatase-2A activity 70 +/- 29% (n = 3, P < 0.038). Thus, the major effects of insulin on phosphatase activity in adipocytes are to increase plasma membrane tyrosine and serine/threonine phosphatase, crude nuclear fraction protein phosphatase-2A, and cytosolic protein phosphatase-1 activities, while inhibiting cytosolic protein phosphatase-2A. Insulin resistance was characterized by reduced insulin-stimulated serine/threonine phosphatase activity in the plasma membrane and low-density microsomes. Specific changes in phosphatase activity may be related to the development of insulin resistance.
