Structural Biology

The Ras signaling cascade acts as a key driver in human colon cancer progression. Among the modules in this pathway, p38γ

(MAPK12) and its specifi c protein tyrosine phosphatase PTPN3 (PTPH1) are critical regulators responsible for Ras oncogenic

activity. However, the molecular basis for their interaction remains elusive. We showed both p38γ and PTPN3 being highly expressed

in late-stage of human colon cancer tissues. Comprehensive structural information on the PTPN3-p38γ interaction is essential

to understand their cooperative activities in Ras-dependent malignancies. Employing an advanced hybrid method integrating

x-ray crystallography, small-angle x-ray scattering (SAXS), chemical cross-linking/mass spectrometry (CX-MS) and hydrogendeuterium

exchange (HDX), we have obtained a unique architecture of the PTPN3-phosphorylated p38γ complex. Moreover, we

showed that PTPN3 binds to unphosphorylated p38γ with micromolar affi nity. Enzyme kinetics studies revealed that the presence

of the PDZ binding motif of p38γ promotes the phosphatase activity of PTPN3. Using small-angle x-ray scattering, we found that

PTPN3 adopts a predominantly compact shape in the absence of p38γ. Aft er complex formation with unphosphorylated p38γ,

we observed an extended conformation that was clearly diff erent from that of the active state complex. Th rough chemical crosslinking

coupled with mass spectrometry, we confi rmed that the PDZ domain binds to the N-lobe region of unphosphorylated

p38γ, thereby forming an end-to-end complex assembly. Overall, our results show that PTPN3 and unphosphorylated p38γ may

form a resting-state complex, suggesting its unexpected function to promote colon cancer progression. Our structural data suggest

the presence of the active-state complex (left ) and the resting-state complex (right) between PTPN3 and p38γ Th e interaction of

PDZ domain in PTPN3 and the ETPL motif in p38γ drives the complex formation. We show that the resting-state complex may

promote oncogenic signaling involved in colon cancer progression.