Over-expression of protein tyrosine phosphatase 1 (PTP1) alters IL-3-dependent growth and tyrosine phosphorylation.
Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.
Gelderloos
J A
JA
Department of Pathology, SUNY at Stony Brook, New York 11794, USA.
Anderson
S M
SM
D000818:Animals; D002455:Cell Division / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D005822:Genetic Vectors; D007377:Interleukin-3 / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D007377:Interleukin-3; D016185:Receptors, Interleukin-3; D017967:Zinc Compounds; C016837:zinc chloride; D011505:Protein-Tyrosine Kinases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases