SH2-Containing protein tyrosine phosphatase-1 (SHP-1) association with Jak2 in UT-7/Epo cells.
We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.
Wu
D W
DW
Department of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, Collegeville, Pennsylvania, USA.
Stark
K C
KC
Dunnington
D
D
Dillon
S B
SB
Yi
T
T
Jones
C
C
Pelus
L M
LM
D000906:Antibodies; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D053614:Janus Kinase 2; D007700:Kinetics; D010455:Peptides; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000276:immunology / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D018909:src Homology Domains
D000906:Antibodies; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D054645:Protein Phosphatase 1; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases