Serine 62-Phosphorylated MYC Associates with Nuclear Lamins and Its Regulation by CIP2A Is Essential for Regenerative Proliferation

An understanding of the mechanisms determining MYC's transcriptional and proliferation-promoting activities in vivo could facilitate approaches for MYC targeting. However, post-translational mechanisms that control MYC function in vivo are poorly understood. Here, we demonstrate that MYC phosphorylation at serine 62 enhances MYC accumulation on Lamin A/C-associated nuclear structures and that the protein phosphatase 2A (PP2A) inhibitor protein CIP2A is required for this process. CIP2A is also critical for serum-induced MYC phosphorylation and for MYC-elicited proliferation induction in vitro. Complementary transgenic approaches and an intestinal regeneration model further demonstrated the in vivo importance of CIP2A and serine 62 phosphorylation for MYC activity upon DNA damage. However, targeting of CIP2A did not influence the normal function of intestinal crypt cells. These data underline the importance of nuclear organization in the regulation of MYC phosphorylation, leading to an in vivo demonstration of a strategy for inhibiting MYC activity without detrimental physiological effects.Peer reviewe

ETS1 Mediates MEK1/2-Dependent Overexpression of Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) in Human Cancer Cells

EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40–80%) in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal −27 to −107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of CIP2A expression and protein phosphatase 2A inhibition

Elucidation of the ATP7B N-Domain Mg2+-ATP Coordination Site and Its Allosteric Regulation

The diagnostic of orphan genetic disease is often a puzzling task as less attention is paid to the elucidation of the pathophysiology of these rare disorders at the molecular level. We present here a multidisciplinary approach using molecular modeling tools and surface plasmonic resonance to study the function of the ATP7B protein, which is impaired in the Wilson disease. Experimentally validated in silico models allow the elucidation in the Nucleotide binding domain (N-domain) of the Mg2+-ATP coordination site and answer to the controversial role of the Mg2+ ion in the nucleotide binding process. The analysis of protein motions revealed a substantial effect on a long flexible loop branched to the N-domain protein core. We demonstrated the capacity of the loop to disrupt the interaction between Mg2+-ATP complex and the N-domain and propose a role for this loop in the allosteric regulation of the nucleotide binding process

Structure and mechanism of Zn^(2+)- transporting P-type ATPases

Zinc is an essential micronutrient for all living organisms. It is required for signalling and proper functioning of a range of proteins involved in, for example, DNA binding and enzymatic catalysis. In prokaryotes and photosynthetic eukaryotes, Zn2+-transporting P-type ATPases of class IB (ZntA) are crucial for cellular redistribution and detoxification of Zn2+ and related elements. Here we present crystal structures representing the phosphoenzyme ground state (E2P) and a dephosphorylation intermediate (E2·P_i) of ZntA from Shigella sonnei, determined at 3.2 Å and 2.7 Å resolution, respectively. The structures reveal a similar fold to Cu^+-ATPases, with an amphipathic helix at the membrane interface. A conserved electronegative funnel connects this region to the intramembranous high-affinity ion-binding site and may promote specific uptake of cellular Zn^(2+) ions by the transporter. The E2P structure displays a wide extracellular release pathway reaching the invariant residues at the high-affinity site, including C392, C394 and D714. The pathway closes in the E2·P_i state, in which D714 interacts with the conserved residue K693, which possibly stimulates Zn^(2+) release as a built-in counter ion, as has been proposed for H^+-ATPases. Indeed, transport studies in liposomes provide experimental support for ZntA activity without counter transport. These findings suggest a mechanistic link between P_(IB)-type Zn^(2+)-ATPases and P_(III)-type H^+-ATPases and at the same time show structural features of the extracellular release pathway that resemble P_(II)-type ATPases such as the sarcoplasmic/endoplasmic reticulum Ca^(2+)-ATPase (SERCA) and Na^+, K^+-ATPase. These findings considerably increase our understanding of zinc transport in cells and represent new possibilities for biotechnology and biomedicine

KIAA1524

Review on KIAA1524, with data on DNA, on the protein encoded, and where the gene is implicated

Oncoprotein CIP2A is stabilized via interaction with tumor suppressor PP2A/B56

Protein phosphatase 2A (PP2A) is a critical human tumor suppressor. Cancerous inhibitor of PP2A (CIP2A) supports the activity of several critical cancer drivers (Akt, MYC, E2F1) and promotes malignancy in most cancer types via PP2A inhibition. However, the 3D structure of CIP2A has not been solved, and it remains enigmatic how it interacts with PP2A. Here, we show by yeast two‐hybrid assays, and subsequent validation experiments, that CIP2A forms homodimers. The homodimerization of CIP2A is confirmed by solving the crystal structure of an N‐terminal CIP2A fragment (amino acids 1–560) at 3.0 Å resolution, and by subsequent structure‐based mutational analyses of the dimerization interface. We further describe that the CIP2A dimer interacts with the PP2A subunits B56α and B56γ. CIP2A binds to the B56 proteins via a conserved N‐terminal region, and dimerization promotes B56 binding. Intriguingly, inhibition of either CIP2A dimerization or B56α/γ expression destabilizes CIP2A, indicating opportunities for controlled degradation. These results provide the first structure–function analysis of the interaction of CIP2A with PP2A/B56 and have direct implications for its targeting in cancer therapy

Solution structure of the N-domain of Wilson disease protein: Distinct nucleotide-binding environment and effects of disease mutations

Wilson disease protein (ATP7B) is a copper-transporting P(1B)-type ATPase that regulates copper homeostasis and biosynthesis of copper-containing enzymes in human tissues. Inactivation of ATP7B or related ATP7A leads to severe neurodegenerative disorders, whereas their overexpression contributes to cancer cell resistance to chemotherapeutics. Copper-transporting ATPases differ from other P-type ATPases in their topology and the sequence of their nucleotide-binding domain (N-domain). To gain insight into the structural basis of ATP7B function, we have solved the structure of the ATP7B N-domain in the presence of ATP by using heteronuclear multidimensional NMR spectroscopy. The N-domain consists of a six-stranded β-sheet with two adjacent α-helical hairpins and, unexpectedly, shows higher similarity to the bacterial K(+)-transporting ATPase KdpB than to the mammalian Ca(2+)-ATPase or Na(+),K(+)-ATPase. The common core structure of P-type ATPases is retained in the 3D fold of the N-domain; however, the nucleotide coordination environment of ATP7B within this fold is different. The residues H1069, G1099, G1101, I1102, G1149, and N1150 conserved in the P(1B)-ATPase subfamily contribute to ATP binding. Analysis of the frequent disease mutation H1069Q demonstrates that this mutation does not significantly affect the structure of the N-domain but prevents tight binding of ATP. The structure of the N-domain accounts for the disruptive effects of >30 known Wilson disease mutations. The unique features of the N-domain provide a structural basis for the development of specific inhibitors and regulators of ATP7B