Abstract
PTEN, a tumor suppressor that is frequently mutated in a wide spectrum of cancers, exerts PI(3,4,5)P3 phosphatase activities that are regulated by its dynamic shuttling between the membrane and cytoplasm. Direct observation of PTEN in the interfacial environment can offer quantitative information about the shuttling dynamics, but remains elusive. Here we show that positively charged residues located in the cα2 helix of the C2 domain are necessary for the membrane localization of PTEN via stable electrostatic interactions in Dictyostelium discoideum. Single-molecule imaging analyses revealed that PTEN molecules moved distances much larger than expected had they been caused by lateral diffusion, a phenomenon we call “hopping.” Our novel single-particle tracking analysis method found that the cα2 helix aids in regulating the hopping and stable-binding states. The dynamically established membrane localization of PTEN was revealed to be essential for developmental processes and clarified a fundamental regulation mechanism of the protein quantity and activity on the plasma membrane.
Highlights
Molecular reactions on the plasma membrane are responsible for various cellular functions
We have discovered that the PTEN protein, which transits between the cytoplasm and membrane, hops along the plasma membrane of living cells
We found that positively charged amino acids in the C2 domain of PTEN, which were reported to be important for its phosphatase activity on the membrane, are required to suppress excessive hopping and stabilize PTEN membrane binding
Summary
Molecular reactions on the plasma membrane are responsible for various cellular functions. Cytoplasmic proteins that possess positively charged patches on their surface due to cationic amino acid residues are electrostatically attracted to the membrane. Such electrostatic interactions provide an essential mechanistic basis for the translocation of cytoplasmic proteins to the membrane, which is usually mediated via domains or motifs of the proteins including the C2 and pleckstrin homology (PH) domains [2,3]. Several regions of PTEN have been identified as necessary for the membrane interaction: the phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)binding motif (PBM) at the N-terminus, the catalytic site and T1 loop in the middle phosphatase domain and cationic patches including the ca helix and CBR3 loop in the C-terminal C2 domain (Fig. 1A) [8]. The localization of PTEN to the membrane is likely regulated via electrostatic interactions with these anionic phospholipids
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