Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bβ

Rui, Liangyou; Gunter, David R.; Herrington, James; Carter‐Su, Christin

doi:10.1128/mcb.20.9.3168-3177.2000

Cited by 60 publications

(68 citation statements)

References 45 publications

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“…2A) (64). As reported previously (60,48), JAK2 is constitutively active when overexpressed in 293T cells and SH2-B␤ greatly enhances JAK2 tyrosyl kinase activity.…”

Section: Pak1 Associates With and Issupporting

confidence: 58%

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JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions

Rider

Shatrova

Feener

et al. 2007

Journal of Biological Chemistry

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The serine-threonine kinase PAK1 is activated by small GTPase-dependent and -independent mechanisms and promotes cell survival. However, the role of tyrosyl phosphorylation in the regulation of PAK1 function is poorly understood. In this study, we have shown that the prolactin-activated tyrosine kinase JAK2 phosphorylates PAK1 in vivo. Wild type, but not kinase-dead, JAK2 directly phosphorylates PAK1 in cells and in an in vitro kinase assay. PAK1 tyrosines 153, 201, and 285 were identified as sites of JAK2 tyrosyl phosphorylation by mass spectrometry and two-dimensional peptide mapping. Mutation of PAK1 tyrosines 153, 201, and 285 to phenylalanines individually or in combination implicated these PAK1 tyrosines in the regulation of PAK1 kinase activity. Tyrosyl phosphorylation by JAK2 significantly increases PAK1 kinase activity, whereas similar phosphorylation of the PAK1 Y153F,Y201F,Y285F mutant has no effect on PAK1 activity. Tyrosyl phosphorylation of wild type PAK1 decreases apoptosis induced by serum deprivation and staurosporine treatment and increases cell motility. In contrast, these parameters are unaltered in the PAK1 Y153F,Y201F,Y285F mutant. Our findings indicate that JAK2 phosphorylates PAK1 at these specific tyrosines and that this phosphorylation plays an important role in cell survival and motility. PAK14 is a member of a conserved family of p21-activated serine-threonine kinases and is important for a variety of cellular functions, including cell morphogenesis, motility, survival, mitosis, and malignant transformation (for review see Refs. 1-3). The emerging roles of PAK1 in the regulation of multiple fundamental cellular processes have directed significant attention toward understanding how PAK1 activity is controlled. Autoinhibition of the PAK1 C-terminal catalytic domain by the N-terminal domain is a key mechanism of PAK1 regulation. Several layers of inhibition, involving dimerization and occupation of the catalytic cleft by contact between the Nand C-terminal domains, keep PAK1 kinase activity in check (4). Autoinhibition of PAK1 occurs in trans, meaning that the inhibitory domain of one PAK1 molecule interacts with the kinase domain of another PAK1 molecule (5). Association of GTP-bound forms of Cdc42 and Rac1 with the PAK1 PBD/ CRIB domain induces conformational changes in the N-terminal domain that no longer support its autoinhibitory function. In addition to Cdc42 and Rac1, PAK1 is activated by the binding of small GTPases, Rac2 and Rac3, as well as TC10, CHP, and Wrich-1 proteins (6 -11). PAK1 is a predominantly cytoplasmic protein, but is activated upon recruitment to the cell membrane. PAK1 membrane localization occurs through interaction with adaptor proteins Nck, Grb2, and PIX, all of which are activated by ligation of growth factor receptors (12-15). Membrane recruitment of PAK1 via adapter proteins and subsequent PAK1 activation may involve phosphorylation at Thr 423 (a site that is also autophosphorylated when PAK1 is activated by Rac1 and Cdc42) by PDK1 (16) or interaction with...

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“…2A) (64). As reported previously (60,48), JAK2 is constitutively active when overexpressed in 293T cells and SH2-B␤ greatly enhances JAK2 tyrosyl kinase activity.…”

Section: Pak1 Associates With and Issupporting

confidence: 58%

“…GST-PAK1 and SH2-B␤ migrate to a similar position after SDS-PAGE. Therefore, to visualize these two proteins on a gel, we used a truncated SH2-B␤-(504 -670) mutant that also increases JAK2 tyrosyl kinase activity (64). Exogenous PAK1 was Tyr phosphorylated in vitro by JAK2 (Fig.…”

Section: Pak1 Associates With and Ismentioning

confidence: 99%

JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions

Rider

Shatrova

Feener

et al. 2007

Journal of Biological Chemistry

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“…The complex from 32Dp210 cells contained P210 Bcr-Abl, Jak2, and the SH2-Bb protein (Figure 4b -d). Carter-Su and colleagues showed that a SH2-Bb SH2 deletion mutant functions as a dominant negative mutant which inhibits ligand-stimulated activation of Stat5B by endogenous receptor and JAK2 in cells responding to growth hormone (Rui et al, 2000). We transfected a similar SH2 mutant, SH2-Bb R555E which has a point mutation in the FLVRES motif of SH2 domain and is defective for binding phosphotyrosine, into 32Dp210 cells under puromycin selection to determine the effects of its expression on c-Myc expression in 32Dp210 cells.…”

Section: Sh2-bb R555e Mutant Decreases Expression Of C-myc Induced Bymentioning

confidence: 99%

“…We reasoned that since Jak2 is required for c-Myc up-regulation, then any action that reduces Jak2 kinase activity would also reduce c-Myc up-regulation in 32Dp210 cells. Dr Carter-Su and colleagues have shown that SH2-Bb expression enhances Jak2 kinase activity (Rui and Carter-Su, 1999;Rui et al, 2000). Therefore, we analysed Jak2/Bcr-Abl complexes from 32Dp210 cells by immunoprecipitation with anti-Jak2 antibody.…”

Section: Sh2-bb R555e Mutant Decreases Expression Of C-myc Induced Bymentioning

confidence: 99%

Jak2 is involved in c-Myc induction by Bcr-Abl

et al. 2002

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We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of BcrAbl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by BcrAbl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bb is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bb. Expression of the SH2-Bb R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in cMyc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein.

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“…We created expression vectors for an enhanced green fluorescent protein chimera in which the fluorescent protein was fused to the amino-terminus of rat JAK2 (EGFP/rJAK2), along with a kinase-inactive control protein (EGFP/ rJAK2(K882E)) [27,28]. Following transient transfection, all three fluorescent chimeric proteins were produced in COS-7 cells and the characteristic green fluorescence was observed microscopically for all recombinant proteins (Figure 1).…”

Section: Microscopic Examination Of Cos-7 Cells Producing Recombinantmentioning

confidence: 99%

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Lee¹,

Duhé²

2006

J Biomed Sci

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SummaryJanus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the ''conventional'' in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2's enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.

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Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bβ

Cited by 60 publications

References 45 publications

JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions

JAK2 Tyrosine Kinase Phosphorylates PAK1 and Regulates PAK1 Activity and Functions

Jak2 is involved in c-Myc induction by Bcr-Abl

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

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