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1.
Cell Signal ; 19(3): 600-9, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17027227

ABSTRACT

Previous work has suggested that the protein tyrosine phosphatase, SHP-2, may act to facilitate angiotensin II (Ang II)-mediated, Jak2-dependent signaling. However, the mechanisms by which this occurs are not known. Here, Ang II-mediated, Jak2-dependent signaling was analyzed in a fibroblast cell line lacking the N-terminal, SH2 domain of SHP-2 (SHP-2(Delta46-110)). While the SHP-2(Delta46-110) cells were capable of activating Jak2 tyrosine kinase, they were unable to facilitate AT1 receptor/Jak2 co-association, STAT activation and subsequent Ang II-mediated gene transcription when compared to wild type control cells. These data therefore suggested that the N-terminal SH2 domain of SHP-2 was acting to recruit Jak2 to the AT1 receptor signaling complex. We found that the N-terminal SH2 domain of SHP-2 binds Jak2 predominantly, but not exclusively at tyrosine 201. Mass spectrometry analysis confirmed that this tyrosine residue is in fact phosphorylated. When this tyrosine was converted to phenylalanine, the ability of Jak2 to activate subsequent downstream signaling events was reduced. In summary, we have identified a novel site of Jak2 tyrosine autophosphorylation; namely, tyrosine 201. Our data suggest that the N-terminal SH2 domain of SHP-2 binds this amino acid residue. The functional consequence of this interaction is to recruit Jak2 to the AT1 receptor signaling complex and in turn promote downstream Jak2-dependent signaling.


Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Janus Kinase 2/metabolism , Protein Tyrosine Phosphatases/metabolism , Receptor, Angiotensin, Type 1/physiology , Signal Transduction , src Homology Domains/physiology , Animals , Blotting, Western , Cell Line , Enzyme Activation , Fibroblasts/metabolism , Genes, Reporter , Glutathione Transferase/metabolism , Intracellular Signaling Peptides and Proteins/chemistry , Janus Kinase 2/genetics , Luciferases/metabolism , Mice , Models, Biological , Models, Molecular , Precipitin Tests , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/chemistry , Recombinant Fusion Proteins/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Tyrosine/metabolism
2.
Mol Cell Biochem ; 265(1-2): 161-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15543946

ABSTRACT

Angiotensin II is a well-known vasoactive peptide, but it can also act as a potent growth factor, partially through activation of the tyrosine kinase Jak2. Activated Jak2 tyrosine phosphorylates and activates members of the Signal Transducers and Activators of Transcription (STAT) family of cytoplasmic transcription factors. Recently, we demonstrated that tryptophan 1020 and glutamic acid 1024 within the Jak2 activation loop are required for Jak2 tyrosine kinase activity. Here, we sought to elucidate the requirement of glutamic acid 1024 for Jak2 function. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative interaction between glutamic acid 1024 and an arginine at position 1113. We generated a series of charge-based substitution mutations at position 1113 and found that conversion of arginine 1113 to glutamic acid, alanine, or lysine prevented Jak2 autophosphorylation. Furthermore, mutation of arginine 1113 prevented the following angiotensin II-dependent processes from occurring: (1) Jak2 tyrosine phosphorylation, (2) Jak2/AT1receptor co-association, (3) STAT1 recruitment to the Jak2/AT1receptor complex, (4) STAT1 tyrosine phosphorylation, and (5) STAT-mediated gene expression. We determined that the interaction between glutamic acid 1024 and arginine 1113 consists of two distinct hydrogen bonds. We conclude that these hydrogen bond interactions are critical for Jak2 kinase function and subsequent angiotensin II-dependent activation of the Jak/STAT signaling pathway.


Subject(s)
Arginine/chemistry , DNA-Binding Proteins/metabolism , Glutamic Acid/chemistry , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/physiology , Trans-Activators/metabolism , Alanine/chemistry , Algorithms , Amino Acid Sequence , Angiotensin II/chemistry , Animals , Blotting, Western , COS Cells , Cell Line , Crystallography, X-Ray , Cytoplasm/metabolism , Gene Expression Regulation , Genes, Dominant , Hydrogen Bonding , Immunoprecipitation , Janus Kinase 2 , Luciferases/metabolism , Lysine/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phosphorylation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , STAT1 Transcription Factor , Signal Transduction , Structure-Activity Relationship , Transfection , Tyrosine/chemistry
3.
Am J Physiol Cell Physiol ; 287(4): C981-91, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15189810

ABSTRACT

Mice lacking a functional Janus kinase 2 (JAK2) allele die embryonically, indicating the mandatory role of JAK2 in basic developmental cellular transcription. Currently, however, the downstream target genes of JAK2 are largely unknown. Here, in vitro conditions were created using a cell line lacking JAK2 expression. Microarray analysis was then used to identify genes that are differentially expressed as a result of the presence, or absence, of JAK2. The data identified 621 JAK2-dependent genes as having at least a twofold change in expression. Surprisingly, these genes did not require ligand-dependent activation of JAK2 but merely its expression in the cell. Thirty-one of these genes were found to have a greater than sevenfold change in expression levels, and a subset of these were further characterized. These genes represent a diverse cluster of ontological functions including transcription factors, signaling molecules, and cell surface receptors. The expression levels of these genes were validated by Northern blot and/or quantitative RT-PCR analysis in both the JAK2 null cells and cells expressing a JAK2-dominant negative allele. As such, this work demonstrates for the first time that, in addition to being a key mediator of ligand-activated gene transcription, JAK2 can perhaps also be viewed as a critical mediator of basal level gene expression.


Subject(s)
Gene Expression Regulation , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Animals , Blotting, Northern , Blotting, Western , Cell Line , Janus Kinase 2 , Mice , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
4.
J Biol Chem ; 279(3): 1956-67, 2004 Jan 16.
Article in English | MEDLINE | ID: mdl-14551204

ABSTRACT

Previous work has shown that inhibition of Jak2 via the pharmacological compound AG490 blocks the angiotensin II (Ang II)-dependent activation of ERK2, thereby suggesting an essential role of Jak2 in ERK activation. However, recent studies have thrown into question the specificity of AG490 and therefore the role of Jak2 in ERK activation. To address this, we reconstituted an Ang II signaling system in a Jak2-/-cell line and measured the ability of Ang II to activate ERK2 in these cells. Controls for this study were the same cells expressing Jak2 via the addition of a Jak2 expression plasmid. In the cells expressing Jak2, Ang II induced a marked increase in ERK2 activity as measured by Western blot analysis and in vitro kinase assays. ERK2 activity returned to basal levels within 30 min. However, in the cells lacking Jak2, Ang II treatment resulted in ERK2 activation that did not return to basal levels until 120 min after ligand addition. Analysis of phosphatase gene expression revealed that Ang II induced mitogen-activated protein kinase phosphatase 1 (MKP-1) expression in cells expressing Jak2 but failed to induce MKP-1 expression in cells lacking Jak2. Therefore, our results suggest that Jak2 is not required for Ang II-induced ERK2 activation. Rather Jak2 is required for Ang II-induced ERK2 inactivation via induction of MKP-1 gene expression.


Subject(s)
Angiotensin II/pharmacology , Cell Cycle Proteins , Immediate-Early Proteins/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Phosphoprotein Phosphatases , Protein Tyrosine Phosphatases/physiology , Protein-Tyrosine Kinases/physiology , Proto-Oncogene Proteins , Cell Line , Cell Nucleus/metabolism , Cytoskeletal Proteins/metabolism , DNA-Binding Proteins/metabolism , Dual Specificity Phosphatase 1 , Enzyme Activation , Humans , Immediate-Early Proteins/genetics , Janus Kinase 2 , Paxillin , Phosphoproteins/metabolism , Phosphorylation , Protein Phosphatase 1 , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Protein Tyrosine Phosphatases/genetics , Receptor, Angiotensin, Type 1/physiology , STAT1 Transcription Factor , Trans-Activators/metabolism , Transcription, Genetic
5.
Mol Cell Biochem ; 241(1-2): 87-94, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12482029

ABSTRACT

Jak2 is a member of the Janus family of tyrosine kinases and is known to be activated by a wide variety of ligands. Here, we sought to identify amino acid residues within Jak2 that are essential for its activation. We provide evidence that glutamic acid 1046 (E1046) is one such residue. Using molecular modeling algorithms of the Jak2 kinase domain, we identified a putative molecular interaction between E1046 and tryptophan 1020 (W1020). Conversion of E1046 to either arginine (E 1046R), alanine (E1046A), aspartic acid (E1046D) or glutamine (E1046Q) abolished Jak2 kinase activity as measured by autophosphorylation assays. Conversion of W1020 to glycine (W1020G) similarly abolished Jak2 kinase activity. Finally, we tested the ability of the E1046R mutant to activate the Jak/STAT signaling pathway in a ligand-dependent signaling system. The ability of angiotensin II to activate the Jak/STAT signaling pathway in cells expressing the E1046R mutant was severely compromised as measured by reduced (1) Jak2 autophosphorylation (2) Jak2 kinase activity (3) AT1/Jak2 co-association (4) Stat1 tyrosine phosphorylation and (5) angiotensin Il-mediated gene transcription. Thus, these studies demonstrate for the first time, the critical role of E1046 in mediating Jak2 activation and its subsequent downstream signaling events.


Subject(s)
Glutamic Acid/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Angiotensin II/metabolism , Animals , Blotting, Western , Cell Line , DNA-Binding Proteins/metabolism , Glutamic Acid/genetics , Janus Kinase 2 , Ligands , Mutagenesis, Site-Directed , Phosphorylation , Protein-Tyrosine Kinases/chemistry , Protein-Tyrosine Kinases/genetics , STAT1 Transcription Factor , Trans-Activators/metabolism , Transcription, Genetic , Tryptophan/genetics , Tryptophan/metabolism
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