Spontaneous and chaperone-assisted metal loading in the active site of protein phosphatase-1.

Protein phosphatase PP1 has two active-site metals (Zn2+/Fe2+) that are essential for catalysis. However, when expressed in bacteria, PP1 has two Mn2+-ions in its active site, indicating that the incorporation of Zn2+/Fe2+ depends on additional eukaryotic component(s). Here, we used purified, metal-deficient PP1 to study metal incorporation. Fe2+ was incorporated spontaneously, but Zn2+ was not. Mn2+-incorporation at physiological pH depended on the co-expression of PP1 with PPP1R2 (Inhibitor-2) or PPP1R11 (Inhibitor-3), or a pre-incubation of PP1 at pH 4. We also demonstrate that PPP1R2 and PPP1R11 are Zn2+-binding proteins but are, by themselves, not able to load PP1 with Zn2+. Our data suggest that PPP1R2 and PPP1R11 function as metal chaperones for PP1 but depend on co-chaperone(s) and/or specific modification(s) for the transfer of associated Zn2+ to PP1.

SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1.

SDS22 forms an inactive complex with nascent protein phosphatase PP1 and Inhibitor-3. SDS22:PP1:Inhibitor-3 is a substrate for the ATPase p97/VCP, which liberates PP1 for binding to canonical regulatory subunits. The exact role of SDS22 in PP1-holoenzyme assembly remains elusive. Here, we show that SDS22 stabilizes nascent PP1. In the absence of SDS22, PP1 is gradually lost, resulting in substrate hyperphosphorylation and a proliferation arrest. Similarly, we identify a female individual with a severe neurodevelopmental disorder bearing an unstable SDS22 mutant, associated with decreased PP1 levels. We furthermore find that SDS22 directly binds to Inhibitor-3 and that this is essential for the stable assembly of SDS22:PP1: Inhibitor-3, the recruitment of p97/VCP, and the extraction of SDS22 during holoenzyme assembly. SDS22 with a disabled Inhibitor-3 binding site co-transfers with PP1 to canonical regulatory subunits, thereby forming non-functional holoenzymes. Our data show that SDS22, through simultaneous interaction with PP1 and Inhibitor-3, integrates the major steps of PP1 holoenzyme assembly.

Protein phosphatase 1: life-course regulation by SDS22 and Inhibitor-3.

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a sizable fraction of protein Ser/Thr dephosphorylation events. It is tightly regulated in space and time through association with a wide array of regulatory interactors of protein phosphatase one (RIPPOs). Suppressor-of-Dis2-number 2 (SDS22) and Inhibitor-3 (I3), which form a ternary complex with PP1, are the first two evolved and most widely expressed RIPPOs. Their deletion causes mitotic-arrest phenotypes and is lethal in some organisms. The role of SDS22 and I3 in PP1 regulation has been a mystery for decades as they were independently identified as both activators and inhibitors of PP1. This conundrum has largely been solved by recent reports showing that SDS22 and I3 control multiple steps of the life course of PP1. Indeed, they contribute to (a) the stabilization and activation of newly translated PP1, (b) the translocation of PP1 to the nucleus, and (c) the storage of PP1 as a reserve for holoenzyme assembly. Preliminary evidence suggests that SDS22 and I3 may also function as scavengers of released or aged PP1 for re-use in holoenzyme assembly or proteolytical degradation, respectively. Hence, SDS22 and I3 are emerging as master regulators of the life course of PP1.

Protein phosphatase-1: dual activity regulation by Inhibitor-2.

Inhibitor-2 (I2) ranks amongst the most ancient regulators of protein phosphatase-1 (PP1). It is a small, intrinsically disordered protein that was originally discovered as a potent inhibitor of PP1. However, later investigations also characterized I2 as an activator of PP1 as well as a chaperone for PP1 folding. Numerous studies disclosed the importance of I2 for diverse cellular processes but did not describe a unifying molecular principle of PP1 regulation. We have re-analyzed the literature on I2 in the light of current insights of PP1 structure and regulation. Extensive biochemical data, largely ignored in the recent I2 literature, provide substantial indirect evidence for a role of I2 as a loader of active-site metals. In addition, I2 appears to function as a competitive inhibitor of PP1 in higher eukaryotes. The published data also demonstrate that several segments of I2 that remain unstructured in the PP1 : I2 complex are in fact essential for PP1 regulation. Together, the available data identify I2 as a dynamic activity-modulator of PP1.

Vimentin expression predicts the occurrence of metastases in non small cell lung carcinomas.

Epithelial-to-mesenchymal transition (EMT) is believed to contribute to tumour invasion. Vimentin expression by carcinoma cells is a largely recognized marker of EMT. This study aimed at examining vimentin expression in non small cell lung carcinomas (NSCLC) by immunohistochemistry to evaluate potential correlations between vimentin expression and the differentiation status, the TNM stage and the outcome of the patients. 295 NSCLC including 164 squamous cell carcinomas (SCC), 108 adenocarcinomas (AC) and 23 other NSCLC carcinomas have been examined by immunohistochemistry. Vimentin was indeed detected in 145 cases (49.2%). It was principally present in isolated tumour cells and invasive clusters, particularly in cells at the tumour/stroma interface. Vimentin expression was significantly more expressed in large cell neuroendocrine, adeno-squamous and sarcomatoid carcinomas than in SCC and AC and was significantly associated with the differentiation status of carcinomas. The follow-up of 193 patients further demonstrated that an extensive expression of vimentin (>50% of tumour cells) was associated with the occurrence of metastases. In conclusion, our data demonstrate that vimentin expression is a frequent event in NSCLC and that its expression can be associated with a lack of differentiation and the occurrence of metastases.