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

Abstract

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.

JAKs go nuclear: Emerging role of nuclear JAK1 and JAK2 in gene expression and cell growth

“…Although ligand treatment did not further increase amounts of JAK protein in the nucleus, levels of tyrosine phosphorylated (activated) JAKs were increased. About the same time, others reported the presence of JAK2 in pancreatic islet cell nuclei (39) and subsequent studies reported JAK2 in nuclei of cultured liver cells and rat liver in vivo (40), as well as COS-7 cells(41). JAK2 was also observed in the nuclei of mature mouse unfertilized oocytes and one-cell and two-cell embryos; interestingly, JAK2 appeared to co-localize with chromosomes (42).…”

Nuclear JAK2

“…11 Nevertheless, it is worth noting that other groups have demonstrated a nuclear localization of JAK2 tagged with either EGFP 10 or smaller epitope tags. 6 Moreover, close inspection of the immunofluorescence (IF) images supplied by Girodon et al ( Figure 1C) 1 demonstrates that the nuclei of HEL cells contain several prominent fluorescent speckles, which were not discussed in their letter.…”

Nuclear JAK2: Form and Function in Cancer

“…The role of cytoplasmic JAK2 has been studied very extensively in the field of cancer research. In hematologic neoplasms such as chronic myelomonocytic leukemia and acute myeloid leukemia JAK2 is constitutively activated because of a mutation (Levine et al,2005; Baxter et al,2005; Tefferi et al,2005; Bina et al,2010), while aberrant JAK activity due to overexpression of wild‐type JAK2 is also associated with a number of solid tumors, including gastric carcinoma, hepatocellular carcinoma, and head and neck squamous cell carcinoma (Lee et al,2006; Fuke et al,2007; Hedvat et al,2009; Kupferman et al,2009). In different experimental models, tumorigenesis is associated with increased activity of JAK2, and functional ablation of JAK2 protects against the onset of various tumors (Sakamoto et al,2009; He and Zhang,2010; Agrawal et al,2011).…”