The intramolecular domain interactions and phosphatase activation mechanisms of Shp2
Shp2 (Src Homology 2 Domain-containing Protein Tyrosine Phosphatase 2) is a pivotal player in various signaling pathways in response to growth factors. It contains two SH2 domains (N-SH2 and C-SH2), a phosphatase catalytic domain and a C-terminal tail containing two tyrosyl phosphorylation sites. While the tyrosine phosphorylation of C-terminal tail has been shown to be critical for the proper Shp2 functions, there is a lack of understanding on how the phosphorylation of these two tyrosine sites regulates the conformational changes and functions of Shp2. Here, we developed a FRET-based Shp2 reporter, a single molecule comprised of the full length human Shp2 concatenated in between a FRET pair, ECFP and YPet. In vitro incubation of Shp2 reporter with PDGFRβ induced a significant FRET increase via intramolecular interaction between C-SH2 and phosphotyrosine 580 (pY580). The phosphotyrosine 542 (pY542) has an adverse position despite a favored surrounding amino acid sequence such that it can only bind to C-SH2 in the absence of pY580. In contrast, pY542 dominates the binding of C-SH2 in mouse embryonic fibroblasts (MEFs). This intramolecular binding is essential for the subsequent effective phosphorylation of Y580, which is then sequestered by Grb2 in cells. These results suggested that the cellular environment can tune and switch the interaction of C-SH2 toward pY542. When the sequences containing Y542 and Y580 were engineered to exchange their positions such that the favored sequence containing Y542 is located at the advantageous position of 580 (Y542’), pY542’ constitutively dominates the binding of C-SH2 both in vitro and in MEFs. This rewiring of intramolecular interactions within Shp2 also caused a reprogramming of downstream signaling events, resulting in a more transient ERK phosphorylation. Therefore, our results indicate that the combination of a favorable sequence with an adverse position can create a delicate balance and plasticity of intramolecular interactions, which can determine the downstream cellular signaling/functions, and may represent a general design principle for functional molecular units tunable by environmental inputs. In addition to the intramolecular interactions we discovered, we also investigate the effect of these interactions on Shp2 phosphatase activity. We found that intramolecular binding between C-SH2 and pY580 doesn’t change Shp2 activity while the binding between C-SH2 and pY542 increases Shp2 activity using phosphatase activity assay. Moreover, phosphatase activity assay also showed that N-SH2 domain can also interact intramolecularly with pY542 or pY580 when C-SH2 is mutated and these interactions increase phosphatase activity. Our FRET reporter also enables us to visualize autodephosphorylation process in vitro. The results with the phosphatase inhibitors and inactive phosphatase mutants showed us that phosphatase activity is not necessary for intramolecular interactions. Even though phosphatase domain doesn’t play a direct role in the intramolecular interactions between SH2 domains and C-tail phosphotyrosines, it can control the level of the phosphotyrosines through its phosphatase activity therefore affect the intramolecular interactions. In conclusion, we have utilized FRET-based reporter to investigate the intramolecular interactions and activation mechanisms of Shp2. We not only showed the tunable intramolecular interactions between C-SH2 and C-tail phosphotyrosines but also established the intrinsic design principles of Shp2 that underlies the tunability. Last but not least, we found the effect of these intramolecular interactions on phosphatase activity and downstream ERK signaling.
Shp2 (Src Homology 2 Domain-containing Protein Tyrosine Phosphatase 2), FRET reporter, Intramolecular interactions, Phosphatase activation, Fluorescent/ Förster resonance energy transfer (FRET)
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign