Central Role of the Threonine Residue within the p+1 Loop of Receptor Tyrosine Kinase in STAT3 Constitutive Phosphorylation in Metastatic Cancer Cells

Abstract

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met p؉1loop 3Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr p؉1loop residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr p؉1loop 3Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK Metp؉1loop with 2B mutation and wild-type EPH members the Thr p؉1loop residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET M918T , nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr p؉1loop residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.Growth factor receptors are transmembrane protein tyrosine kinases playing critical roles in biological processes, including proliferation, survival, and migration. The core region of the kinase domain, comprising the catalytic loop, the activation loop, and the pϩ1 loop, is well conserved in receptor tyrosine kinases (RTKs) of different species. RTKs with different types of mutations within these three loops have been associated with developmental disorders and cancer. The point mutation (ATG3ACG), resulting in replacement of methionine with threonine within the pϩ1 loop, is associated with aggressive tumors. RET is an RTK that can activate a variety of signaling pathways, including the RAS/ERK, PI3K/AKT, and phospholipase C␥ pathways and plays an important role in neuron survival or differentiation (11). RET with a Met pϩ1loop 3Thr substitution (RET M918T ) is associated with the multiple endocrine neoplasia 2B type (MEN-2B) syndrome; this substitution is defined as the 2B mutation (11). In MEN-2B patients, the tumors derived from thyroid C cells are often more aggressive than C-cell tumors that develop in MEN-2A patients who carry mutations in the extracellular domain of RET (11,22). Similarly, the 2B mutation in HGF receptor MET (Met M1268T ) has been identified in metastatic renal carcinomas (10,24). Introduction of the 2B mutation in other RTKs, such as RON and epidermal growth factor receptor (EGFR), caused transformation of NIH 3T3 cells with high metastatic potential (20,23). Although the 2B mutation enhanced kinase activity and such a mutation has been suspected as a g...

Current concepts in RET-related genetics, signaling and therapeutics

“…By contrast, the FMTC-associated mutants RETY791F and RETS891A implicate SRC and JAKs in constitutive activation of STAT3 [33]. Moreover, Yuan et al [34] have demonstrated that RET-MEN2B mutants interact with and activate STAT3 more strongly than do RET-MEN2A mutants. These data support previous findings showing that RET-MEN2B mutants are specifically associated with SRC, of which STAT3 is one of the best-characterized targets [35].…”

Novel targeted therapeutics for MEN2

“…On the contrary, activating point mutations targeting the kinase domain, such as RET Y791F and RET S891A (FMTC) implicate JAKs and SRC kinases in the constitutive activation of STAT3 (Plaza Menacho et al 2005). In another study, oncogenic RET M918T (MEN2B) was shown to interact with and activated STAT3 more strongly than RET C634R (MEN2A) (Yuan et al 2004). In the same line, oncogenic RET enhanced in cells the phosphorylation levels of FAK activation loop Y576/577 phosphorylation compared to wild-type RET, and kinase domain mutants (RET M918T-MEN2B and RET V804M, a FMTC-associated drug-resistant mutant) induced a more robust phosphorylation than mutants targeting the extracellular domain (e.g., RET C634R, MEN2A).…”

“…RET MEN2B variants, in particular RET M918T, show not only an altered catalytic activity but also an altered substrate specificity because they preferentially phosphorylate substrates that, contrary to wild-type RET, are usually preferred by cytoplasmic tyrosine kinases such us FAK and SRC (Kato et al 2002, Encinas et al 2004, PlazaMenacho et al 2011. The transcription factor STAT3 is one such example (Yuan et al 2004). In addition, RET-MEN2B mutants seem to lack dependency on activation loop Y905 for both cells transformation and signaling (Iwashita et al 1996.…”

Combination of mRNA and protein microarray analysis in complex cell profiling

“…The cell can control the rates of degradation or synthesis for a given protein, and there is heterogeneity even among proteins that have similar functions [25]. Antibody microarrays have been widely used to profile proteins that define specific cancer types [26][27][28], and to detect post-translational modifications in malignant processes [29,30]. The approach of combining antibody microarrays and cDNA microarrays for complex molecular cell profiling was used previously to look for differences in the expression patterns of muscle cells from a spinal muscular atrophy patient [31].…”