A Catalytically Active Jak2 Is Required for the Angiotensin II-dependent Activation of Fyn

Abstract

Recent work with interleukins has shown a convergence of tyrosine phosphorylation signal transduction cascades at the level of the Janus and Src families of tyrosine kinases. Here we demonstrate that activation of the seven-transmembrane AT 1 receptor by angiotensin II induces a physical association between Jak2 and Fyn, in vivo. This association requires the catalytic activity of Jak2 but not Fyn. Deletion studies indicate that the region of Jak2 that binds Fyn is located between amino acids 1 and 240. Studies of the Fyn SH2 and SH3 domains demonstrate that the SH2 domain plays the primary role in Jak2/Fyn association. Not surprisingly, this domain shows a marked preference for tyrosinephosphorylated Jak2. Surface plasmon resonance estimated the dissociation equilibrium constant (K d ) of this association to be 2.36 nM. Last, in vivo studies in vascular smooth muscle cells show that, in response to angiotensin II, Jak2 activation is required for Fyn activation and induction of the c-fos gene. The significance of these data is that Jak2, in addition to serving as a critical angiotensin II activated signal transduction kinase, also functions as a docking protein and participates in the activation of Fyn by providing phosphotyrosine residues that bind the SH2 domain of Fyn.The Jak family of nonreceptor tyrosine kinases includes Jak1, Jak2, Jak3, and Tyk2. Each protein is approximately 130 kDa and contains seven conserved Jak homology domains (JH1 to JH7) (1, 2). The Jak kinases induce gene regulation through the signal transducers and activators of transcription. Unlike almost all other protein-tyrosine kinases, members of the Jak family bear no SH2 1 or SH3 domains. In contrast to the Jaks, members of the Src family of protein-tyrosine kinases are approximately 55-62 kDa in mass and do possess SH2 and SH3 domains. There are nine known members of the Src kinase family. While the expression of most members is restricted to hematopoietic cells, Fyn and Src are widely expressed by many cell types. There appears to be some functional redundancy of these two proteins, since knockout mice lacking either gene are born alive, while disruption of both the fyn and src alleles results in embryonic lethality (3-5). This redundancy is also seen in the activation of similar signaling pathways by Fyn and Src (6).Recent studies have demonstrated various levels of crosstalk between Jak2 and other signaling pathways. For example, activation of gp130 by the hematopoietic cytokine, interleukin-11, induces protein complex formation between the Jak and Src family tyrosine kinases. Specifically, treatment of 3T3-L1 cells with interleukin-11 leads to a transient complex of Grb2, Jak2, and Fyn (7). Subsequent studies by Yang et al. demonstrated an interleukin-11-dependent association of Jak2 with other signaling molecules including protein phosphatase 2A, phosphatidylinositol-3-kinase, and the Src family kinase Yes (8). To date, the regions that mediate these interactions, as well as the hierarchy of the signal transduction cascades, hav...

Jak2 Tyrosine Kinase Mediates Oxidative Stress-induced Apoptosis in Vascular Smooth Muscle Cells

“…RASM-control cells were created by transfecting primary RASM cells with a neomycin-resistant cassette, whereas RASM-DN cells were created by stably transfecting RASM cells with a Jak2 dominant negative mutant in order to inhibit the function of endogenously expressed Jak2. These cells have been characterized previously (14). To determine whether expression of the Jak2 dominant negative could inhibit Jak2 tyrosine phosphorylation in response to hydrogen peroxide, the two cell types were treated with 0.2, 0.5, or 1 mM hydrogen peroxide for 0, 5, or 10 min.…”

“…Cell Culture-Creation and characterization of the RASM-control and RASM-DN cells has been described previously (14). These cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and 10% fetal bovine serum and growth-arrested in serum-free Dulbecco's modified Eagle's medium for 48 h prior to the experiments.…”

“…RASM-DN cells were created by stably transfecting RASM cells with a Jak2 dominant negative mutant to inhibit the function of endogenously expressed Jak2. The creation and characterization of these cells has been described previously (14).…”

Review: Signal transduction mechanisms of the angiotensin II type AT1-receptor: looking beyond the heterotrimeric G protein paradigm

“…To determine this, a vascular smooth muscle cell line was generated which overexpressed a catalytically-inactive form of Jak2. 38 It was previously demonstrated that inhibition of the Jak2 kinase domain, either by pharmacological means or by a point mutation in the kinase domain, prevented Jak2 from binding the AT 1 -receptor. 39 Since the modified smooth muscle cell line expressed the catalytically inactive form of Jak2, Ang II treatment would not induce AT 1 /Jak2 physical co-association.…”

“…Using this system, it was demonstrated that STAT1 tyrosine phosphorylation was severely compromised in cells that expressed the inactive form of Jak2. 38 To determine whether the failure of STAT1 tyrosine phosphorylation resulted in the specific loss of gene transcription within the nucleus, the Ang II-dependent accumulation of c-fos mRNA was examined. Ang II induces the expression of several early growth response genes including c-fos.…”