Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.
Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the
KLK3
(
PSA
) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.
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