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. 2006 Jun;26(11):4052-62.
doi: 10.1128/MCB.01591-05.

Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor

Anna M Mazurkiewicz-Munoz  1 , Lawrence S Argetsinger, Jean-Louis K Kouadio, Allan Stensballe, Ole N Jensen, Joel M Cline, Christin Carter-Su
Affiliations

Phosphorylation of JAK2 at serine 523: a negative regulator of JAK2 that is stimulated by growth hormone and epidermal growth factor

Anna M Mazurkiewicz-Munoz et al. Mol Cell Biol. 2006 Jun.

Abstract

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling for multiple receptor tyrosine kinases and several G-protein-coupled receptors. In this study, phosphopeptide affinity enrichment and mass spectrometry identified serine 523 (Ser523) in JAK2 as a site of phosphorylation. A phosphoserine 523 antibody revealed that Ser523 is rapidly but transiently phosphorylated in response to growth hormone (GH). MEK1 inhibitor UO126 suppresses GH-dependent phosphorylation of Ser523, suggesting that extracellular signal-regulated kinases (ERKs) 1 and/or 2 or another kinase downstream of MEK1 phosphorylate Ser523 in response to GH. Other ERK activators, phorbol 12-myristate 13-acetate and epidermal growth factor, also stimulate phosphorylation of Ser523. When Ser523 in JAK2 was mutated, JAK2 kinase activity as well as GH-dependent tyrosyl phosphorylation of JAK2 and Stat5 was enhanced, suggesting that phosphorylation of Ser523 inhibits JAK2 kinase activity. We hypothesize that phosphorylation of Ser523 in JAK2 by ERKs 1 and/or 2 or other as-yet-unidentified kinases acts in a negative feedback manner to dampen activation of JAK2 in response to GH and provides a mechanism by which prior exposure to environmental factors that regulate Ser523 phosphorylation might modulate the cell's response to GH.

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Figures

FIG. 1.
FIG. 1.
JAK2 is phosphorylated on serine 523. JAK2 was isolated from Sf9 cells infected with baculovirus containing the cDNA for murine JAK2. JAK2 was digested with trypsin and analyzed by nanoelectrospray quadrupole time of flight tandem mass spectrometry. The doubly charged ion corresponding to T514NGISDVQI[pS]PTLQR528and the Y-ion series corresponding to the C-terminal peptide ion fragments are indicated. The increment corresponding to the phosphorylated serine (pS) is indicated and corresponds to the molecular weight of a phosphorylated serine residue.
FIG. 2.
FIG. 2.
GH promotes the phosphorylation of JAK2 at serine 523. (A) 293T cells transfected with cDNA (1 μg) encoding either JAK2 (lane 1) or JAK2 S523A (lane 2) were lysed. The lysates were immunoblotted (IB) with αpSer523 JAK2 (upper panel) or αJAK2 (lower panel) (n = 4). wt, wild type. (B) 3T3-F442A cells were treated with vehicle or with 500 ng GH/ml for 5, 10, or 15 min. The cells were lysed, and lysates were immunoblotted (IB) with αpS523 JAK2 (top panel), αpY1007/1008 JAK2 (second panel), αpY570 JAK2 (third panel), or αJAK2 (bottom panel). (C) Replicates for the experiment shown in panel B were normalized to levels of JAK2. Means ± standard errors of the means are shown for n = 6. (D) 3T3-F442A cells were treated with vehicle (lanes 1 and 5) or with 500 ng GH/ml (lanes 2 to 4 and 6 to 8). Cells were lysed, and lysates were incubated without (lanes 1 to 4) or with (lanes 5 to 8) 0.7 U pp2A at 37°C for 60 min. Cell lysates were immunoblotted with either αpS523 JAK2 (top panel) or αJAK2 (bottom panel). The migration of JAK2 is indicated. (E) Results from the experiment shown in panel D were quantified and normalized to levels of JAK2.
FIG. 3.
FIG. 3.
MEK1 inhibitor UO126 inhibits GH-stimulated phosphorylation of serine 523 in JAK2. (A) 3T3-F442A cells were incubated with vehicle (lanes 1 to 3) or with 10 μM UO126 (lanes 4 to 6) for 30 min. Cells were then treated with vehicle (lanes 1 and 4) or with 500 ng GH/ml for 5 or 10 min (lanes 2 and 3 and lanes 5 and 6). Cell lysates were immunoblotted (IB) with αpS523 JAK2 (top panel), αJAK2 (second panel), αpErk (third panel), or αErk (bottom panel). The migration of JAK2 and the ERKs is indicated. (B) Replicates for the experiment shown in panel A were quantified and normalized to levels of JAK2. Means ± standard errors of the means are shown for n = 4.
FIG. 4.
FIG. 4.
MEK1 inhibitor UO126 inhibits PMA-stimulated phosphorylation of JAK2 on serine 523. (A) 3T3-F442A cells were pretreated with DMSO (lanes 1 to 3) or with 10 μM UO126 in DMSO (lanes 4 and 5) for 30 min prior to stimulation with vehicle (lane 1) or with 1 μM PMA for the indicated times (lanes 2 to 5). Cells were lysed, and the lysates were immunoblotted (IB) with αpS523 JAK2 (top panel), αJAK2 (middle panel), or αpErk (bottom panel). The migration of JAK2, a nonspecific band (ns), and the ERKs is indicated. (B) Replicates for the experiment shown in panel A were quantified and normalized to levels of JAK2. Means and the range are shown for n = 2.
FIG. 5.
FIG. 5.
EGF promotes the phosphorylation of JAK2 at serine 523. 3T3-F442A cells were stimulated with vehicle or with 125 ng EGF/ml for 5, 10, 15, or 30 min. Cells were lysed, and lysates were immunoblotted with αpS523 JAK2 (top panel), αpY1007/1008 JAK2 (middle panel), or αJAK2 (bottom panel), n = 2. The migration of JAK2 and a nonspecific band (ns) is indicated.
FIG. 6.
FIG. 6.
Mutation of serine 523 to alanine in JAK2 elevates JAK2 kinase activity. (A) 293T cells were transfected with cDNA (0.5 μg) encoding JAK2 (lane 1) or JAK2 S523A (lane 2). Cell lysates (top panel) or αJAK2 immunoprecipitates (bottom panel) were immunoblotted with αJAK2. The migration of JAK2 is indicated. wt, wild type. (B) Immunoprecipitated JAK2 or JAK2 S523A was subjected to an in vitro kinase assay for 20, 40, and 60 min using 50 μM ATP and 500 μM of a peptide containing tyrosine 699 of Stat5 as substrate. (C) Immunoprecipitated JAK2 or JAK2 S523A was subjected to an in vitro kinase assay at Stat5 peptide substrate concentrations between 0 and 500 μM. The reaction velocity was determined by averaging the rate of 32P incorporation into the Stat5 peptide at 40 and 60 min, n = 2. (D) COS-7 cells were transfected with cDNAs encoding GH receptor (100 ng), Stat5b (200 ng), and either JAK2 (20 ng) or JAK2 S523A (20 ng). Cells were incubated in serum-free medium overnight and resolved on a 5 to 12% gradient gel. Cell lysates were immunoblotted with αpStat5b, αStat5b, or αJAK2 as indicated, n = 2.
FIG. 7.
FIG. 7.
Mutation of serine 523 to alanine in JAK2 enhances GH-dependent phosphorylation of JAK2 and Stat5b. (A) COS-7 cells were transfected with cDNAs encoding GH receptor (100 ng), Stat5b (200 ng), and either JAK2 (10 ng) (lanes 1 to 6) or JAK2 S523A (10 ng) (lanes 7 to 12). Cells were treated with vehicle or 500 ng GH/ml for the indicated times. Cell lysates were immunoprecipitated with αJAK2 and immunoblotted with αPY (top panel) or αJAK2 (second panel). Cell lysates were blotted with αpStat5b (third panel) or αStat5b (bottom panel), n = 2. The migration of JAK2 and Stat5b is indicated. wt, wild type. (B and C) The intensities of the bands corresponding to phosphorylated JAK2 and phosphorylated Stat5b were quantified, normalized for levels of JAK2 and Stat5, and plotted as the fraction of maximum phosphorylation detected with αPY (B) and with αpStat5 (C).
FIG. 8.
FIG. 8.
Pretreatment with EGF inhibits GH-dependent activation of JAK2. 3T3-F442A cells were treated with 125 ng EGF/ml as indicated or treated with 125 ng EGF/ml or vehicle for 30 min and then treated with vehicle or 50 ng GH/ml as indicated. Cell lysates were blotted with αpY1007/1008 JAK2, αPY, or αJAK2. The intensities of the bands corresponding to phosphorylated JAK2 were quantified, normalized to levels of JAK2, and graphed as the fraction of maximum phosphorylation detected with αpY1007 (A) and αPY (B) (error bars denote ranges of values, n = 2).
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