ERBB4/HER4 potentiates STAT5A transcriptional activity by regulating novel STAT5A serine phosphorylation events.
The epidermal growth factor receptor family member ERBB4 is required for mammary gland development and lactation. ERBB4 activities in the breast are mediated through the signal transducer and activator of transcription (STAT) family member STAT5A, and ERBB4 directly activates STAT5A, in part, through phosphorylation of STAT5A at the regulatory Tyr-694. Here we show that STAT5A regulation by ERBB4 is also mediated through STAT5A serine phosphorylation. Using a reverse-phase high performance liquid chromatography tandem mass spectrometry analysis of proteolytically digested STAT5A coexpressed with ERBB4, we identified STAT5A serine phosphorylations at the previously described Ser-779 and at the novel Ser-127/Ser-128. Immunohistochemistry of wild-type and ERBB4-null mammary glands at late pregnancy showed that ERBB4 expression was required for STAT5A phosphorylation at Ser-779. Independent serine-to-alanine residue substitutions in full-length STAT5A revealed that although STAT5A Ser-779 phosphorylation was dispensable for phosphorylation of STAT5A at Tyr-694 and subsequent DNA binding, Ser-779 was required to stabilize an interaction with ERBB4 and mediate ERBB4-induced STAT5A stimulation of gene expression. STAT5A Ser-127/Ser-128, on the other hand, was required for ERBB4-induced phosphorylation of Tyr-694, whereas Ser-779 and as yet unidentified tyrosine residues were phosphorylated in the absence of Ser-127/Ser-128. In addition, STAT5A S127A/S128A remained associated with ERBB4 but failed to bind DNA or activate transcription in response to ERBB4 coexpression. Our studies demonstrate that phosphorylation of STAT5A at Ser-127/Ser-128 and Ser-779 are obligatory events regulating ERBB4-mediated activation of STAT5A.
Clark
Diane E
DE
Department of Biochemistry, Tulane University Health Sciences Center, Tulane Cancer Center, New Orleans, Louisiana 70112-2669, USA.
Williams
Christopher C
CC
Duplessis
Tamika T
TT
Moring
Kimberly L
KL
Notwick
Amy R
AR
Long
Weiwen
W
Lane
William S
WS
Beuvink
Iwan
I
Hynes
Nancy E
NE
Jones
Frank E
FE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D066246:ErbB Receptors / Q000502:physiology*; D005260:Female; D007150:Immunohistochemistry; D008321:Mammary Glands, Animal / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008894:Milk Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011247:Pregnancy; D066247:Receptor, ErbB-4; D050799:STAT5 Transcription Factor; D012694:Serine / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050799:STAT5 Transcription Factor; C494093:Stat5a protein, mouse; D015534:Trans-Activators; D012694:Serine; D066246:ErbB Receptors; C587690:Erbb4 protein, mouse; D066247:Receptor, ErbB-4