Differential nuclear localization and kinase activity of alternative ErbB4 intracellular domains

Abstract

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene b-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.

Suppression of breast cancer cell growth by a monoclonal antibody targeting cleavable ErbB4 isoforms

“…The expression plasmids pcDNA3.1ErbB4JM-aCYT-1, pcDNA3.1ErbB4JM-aCYT-2, pcDNA3.1ErbB4JM-bCYT-2, pcDNA3.1ErbB4JM-aCYT-2-HA and pcDNA3.1ErbB4JM-bCYT-2-HA (Ma¨a¨tta¨et al, 2006;Sundvall et al, 2007) were used to transiently express ErbB4 isoforms with or without C-terminal HA epitope tags. To generate kinase-dead ErbB4 construct, the putative ATP-binding site within the kinase domain of ErbB4 was mutated (K751R) in pcDNA3.1ErbB4JM-aCYT-2 using a site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) to produce pcDNA3.1ErbB4JM-aCYT-2-K751R.…”

“…To study ErbB4 ubiquitination, COS-7 cells were transiently transfected with plasmids encoding full-length JM-a CYT-1 or JM-a CYT-2 together with Flag-tagged ubiquitin (Katz et al, 2002), stimulated for 10, 30 or 120 min with or without 2 mg/ml mAb 1479, and analysed by ErbB4 immunoprecipitation followed by anti-Flag western blotting, as previously described (Sundvall et al, 2007).…”

Protein Inhibitor of Activated STAT3 (PIAS3) Protein Promotes SUMOylation and Nuclear Sequestration of the Intracellular Domain of ErbB4 Protein

“…Cell Fractionation-Cytosolic and nuclear fractions were prepared with a NE-PER kit (Thermo Scientific) as described previously (20) and analyzed by Western blotting with anti-HA, anti-FLAG, anti-ErbB4 (E200, Abcam), and anti-PIAS3 (sc-46682) as indicated. Antibodies against Lamin B (sc-6217, Santa Cruz Biotechnology) and Mek1/2 (Cell Signaling) were used to control the fractionation.…”

Developmental pruning of excitatory synaptic inputs to parvalbumin interneurons in monkey prefrontal cortex

“…Moreover, ErbB4 transcripts are alternatively spliced at two loci, generating four different splice variants with distinct downstream signaling pathways (21). Although their functional consequences are not known in PV interneurons, ErbB4 splice variants display different levels of tyrosine kinase activity and exert different physiological effects in nonneuronal cells (22)(23)(24), suggesting that the effect of ErbB4 signaling could be modulated by alternative splicing. Thus, shifts in the expression level and/or splicing of ErbB4 in PV interneurons during adolescence might function as a developmental switch regulating the pruning of excitatory synapses on these neurons.…”