George S. Campbell

Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase

Growth hormone receptor (GHR) forms a complex with a tyrosine kinase, suggesting involvement of a ligand-activated tyrosine kinase in intracellular signaling by growth hormone (GH). Here we identify JAK2, a nonreceptor tyrosine kinase, as a GHR-associated tyrosine kinase. Immunological approaches were used to establish GH-dependent complex formation between JAK2 and GHR, activation of JAK2 tyrosine kinase activity, and tyrosyl phosphorylation of both JAK2 and GHR. The JAK2-GHR and JAK2-erythropoietin receptor interactions described here and in the accompanying paper provide a molecular basis for involvement of tyrosyl phosphorylation in physiological responses to these ligands and suggest a shared signaling mechanism among members of the cytokine/hematopoietin receptor family.

Constitutive activation of JAK1 in Src-transformed cells.

We have previously found that the ignal ransducer and ctivator of ranscription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was assessed using autophosphorylation assays. In anti-JAK1 immunoprecipitates from v-Src-transformed NIH3T3 cells, a protein with the same migration as JAK1 showed substantially increased levels of 32P incorporation compared to immunoprecipitates from non-transformed cells. Similar results were obtained using anti-JAK2 immunoprecipitates; however, the level of JAK2 tyrosyl phosphorylation and 32P incorporation in anti-JAK2 immunoprecipitates were markedly lower than in anti-JAK1 immunoprecipitates. We conclude that JAK1, and possibly JAK2, are constitutively activated in Src-transformed cells, raising the possibility that Janus family kinases contribute to the constitutive activation of Stat3 previously observed in these cells and/or other properties of Src-transformed cells.

Serotonin Stimulates Protein Tyrosyl Phosphorylation and Vascular Contraction via Tyrosine Kinase

Serotonin (5-HT, 5-hydroxytryptamine) is a mitogen in vascular smooth muscle and vascular reactivity to 5-HT is significantly enhanced in hypertension and atherosclerosis. We have tested the hypothesis that tyrosine kinases, enzymes important for mitogenesis, may play a role in 5-HT-induced vascular smooth muscle contractility. Helical strips of rat carotid artery and aorta denuded of endothelium were mounted in tissue baths for measurement of contractile force. The tyrosine kinase inhibitor genistein (5 x 10(-6) M) decreased the potency of 5-HT approximately 4-fold and reduced maximal contraction to 5-HT in carotid arterial strips denuded of endothelium (58% control). Genisteins inactive congener daidzein (5 x 10(-6) M) did not reduce maximal contraction to 5-HT in carotid arteries but did shift the 5-HT concentration response curve 3-fold to the right. Tyrphostin 23 (5 x 10(-5) M), another tyrosine kinase inhibitor, decreased the potency of 5-HT 4-fold and reduced the maximal contraction to 5-HT in the carotid artery (10% control). Contractions induced by phorbol-12,13-dibutyrate (10(-9) to 10(-5) M) were not reduced or shifted by either tyrosine kinase inhibitor, indicating that phorbolester-sensitive protein kinase C isoforms were not affected. KCl-induced contraction was shifted 2-fold and the maximum significantly inhibited by tyrphostin 23 (38.6% control) but not genistein or daidzein, indicating that tyrphostin 23 but not genistein may inhibit voltage-gated calcium channels to reduce contractility. Western blot analysis using antiphosphotyrosine antibody confirmed that 5-HT produced a time- and concentration-dependent increase in the phosphotyrosine immunoreactivity of a 42-kD protein in cultured aortic smooth muscle cells. Lysate immunoprecipitation with an antimitogen-activated-protein (MAP)-kinase antibody indicated that the 42-kD protein was most likely a MAP kinase. 5-HT (10(-5) M) stimulated contraction and increased antiphosphotyrosine immunoreactivity in whole aorta mounted in tissue baths. Importantly, aortic contraction to 5-HT was shifted (5-fold rightward) and reduced (69% control) by genistein but not daidzein. These findings demonstrate that (1) tyrosine kinase activation may partially mediate contractility to 5-HT in arterial smooth muscle, (2) tyrphostin 23 is somewhat nonselective and (3) 5-HT stimulates tyrosine kinase as documented by increased tyrosyl phosphorylation of proteins in cultured aortic smooth muscle cells and aortic tissue in active contraction of 5-HT. These findings have significant implications not only in understanding a novel pathway of 5-HT signal transduction but also in vascular diseases in which growth and/or contractility to 5-HT is increased (e.g. hypertension, atherosclerosis).

Regulation of Glucose Transport and c-fos and egr-1 Expression in Cells with Mutated or Endogenous Growth Hormone Receptors

To identify mechanisms by which GH receptors (GHR) mediate downstream events representative of growth and metabolic responses to GH, stimulation by GH of c-fos and egr-1 expression and glucose transport activity were examined in Chinese hamster ovary (CHO) cells expressing mutated GHR. In CHO cells expressing wild-type GHR (GHR1–638), GH stimulated the expression of c-fos and egr-1, and stimulated 2-deoxyglucose uptake, responses also mediated by endogenous GHR in 3T3-F442A cells. Deletion of the proline-rich box 1 of GHR (GHRΔP) abrogated all of these responses to GH, indicating that box 1, a site of association of GHR with the tyrosine kinase JAK2, is crucial for these GH-stimulated responses. As the C-terminal half of the cytoplasmic domain of GHR is required for GH-stimulated calcium flux and for stimulation of spi-2.1 transcription, GHR lacking this sequence (GHR1–454) were examined. Not only did GHR1–454 mediate stimulation of c-fos and egr-1 expression and 2-deoxyglucose uptake, but they also media...

The Identification of JAK2 Tyrosine Kinase as a Signaling Molecule for Growth Hormone

Abstract The intracellular pathways by which the binding of growth hormone (GH) to its receptor elicits its diverse effects have eluded investigators for many years. Studies showing that GH rapidly stimulates tyrosyl phosphorylation of cellular proteins, and that tyrosine kinase activity co-purifies with GH-GH receptor complexes, led us to hypothesize that activation by GH of a receptor-associated tyrosine kinase is an important early, and perhaps, initiating step in signal transduction by GH. Here, we review the work identifying JAK2 as a GH receptor-associated tyrosine kinase that is rapidly activated by ligand binding.

Regulation and Genetics of Amino Acid Transport

The major transport systems for the uptake of neutral amino acids in mammalian cells have been designated A, ASC, and L.’,’ The transport systems and their regulation have been characterized in Chinese hamster ovary (CHO) System A is sodium-dependent, subject to trans-inhibition, and serves for the uptake of amino acids with short, polar, or linear side chains. System ASC is also sodium dependent and has a strong preference for alanine, serine, and cysteine. In the C H O cell, the ASC system shows a somewhat broader specificity than that found in the Ehrlich cell.* Unlike System A, System ASC does not tolerate N-methylated substrates such as 2-methylaminoisobutyric acid (MeAIB). System L is sodium-independent and serves for the uptake of branched-chain and aromatic amino acids. We operationally define the systems as follows: System A can be represented by the sodium-dependent uptake of 0.2 m M 2-arninoisobutyric acid (AIB) that is inhibited by 25 m M MeAIB; System ASC, the sodium-dependent uptake of 0.2 m M L-alanine that is not inhibited by 25 m M MeAIB; and System L, the sodium-independent uptake of 0.2 m M L-leucine that is inhibited by 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH).3 Although the systems have a preferred set of substrates, they do have overlapping specificities. FIGURE 1 shows the contributions of these systems to the uptake of individual amino acids in CHO-K1 cells. This overlap makes the study of transport systems in isolation difficult. The availability of mutations in one or more of the transport systems would greatly facilitate the study of the function and regulation of the transport systems. We are currently combining genetic approaches with kinetic studies using C H O cells because of the relative ease with which mutants can be obtained from these cells. Furthermore, C H O cells can be used to form interspecies hybrids with human cells.’ The hamster-human hybrid cells preferentially segregate the human chromosomes, permitting the assignment of a phenotype to a particular chromosome. In the present study, we have isolated and characterized C H O mutants defective in the regulation of System L6 and mutants with reduced System L transport activity. We have also used hamster-human hybrids to map System L transport activity to human chromosome 20.’