Phosphorylation of a serine/proline-rich motif in oxysterol binding protein-related protein 4L (ORP4L) regulates cholesterol and vimentin binding

Abstract

The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S 762 SPSSPSS 769 ) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [ 32 P]PO 4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.

Mechanical and Non‐Mechanical Functions of Filamentous and Non‐Filamentous Vimentin

“…[ 31 ] Phosphorylation of the oxysterol binding protein 4L (ORP4L) leads to its binding to vimentin and facilitates cholesterol extraction from membranes, further implicating vimentin in lipid droplet formation and processing. [ 32 ]…”

Profiling the Human Phosphoproteome to Estimate the True Extent of Protein Phosphorylation

“…4d & 4e) was not as strong as the enrichment of Pro at +1 position (Fig. 4a & 4b), and not as well cited as Pro in relation to phosphorylation (Hall and Vulliet, 1991;Keshwani et al, 2015;Lu, Liou and Zhou, 2002;Pietrangelo and Ridgway, 2019). As a result, we only relied on Pro enrichment at +1 positions to maximise the accuracy of FDR estimations within the sets of Ser and Thr sites.…”

“…Therefore, the enrichment of Pro around highly likely Ser and Thr phosphosites suggests that this feature, amongst others, can be used as a differentiating characteristic for phosphosites compared to nonphosphosites. Furthermore, multiple previous reports specifically highlight the importance of Pro in the mechanism of phosphorylation for families of kinases such as the cyclin-dependent kinases, Mitogen Activated Protein Kinases and, more recently, the centrosomal kinase PLK4 (Byrne et al, 2020;Hall and Vulliet, 1991;Johnson et al, 1998;Keshwani et al, 2015;Lu, Liou and Zhou, 2002;Pietrangelo and Ridgway, 2019;Songyang et al, 1994). Consequently, there is a high prevalence of Pro in numerous phosphorylation motif sequences as part of Ser/Thr-Pro combinations (Amanchy et al, 2007;Sugiyama, Imamura and Ishihama, 2019).…”

Endoplasmic reticulum–plasma membrane contacts: Principals of phosphoinositide and calcium signaling

“…Moreover, ORP4L is proposed to bind PI(4,5)P 2 , Gαq, and PLCβ3 to promote PLCβ3 activity necessary for leukemia stem cell growth and survival [48]. ORP4L phosphorylation enhances cholesterol binding, possibly at the expense of PI(4,5)P 2 binding [49], providing a potential regulatory mechanism for ORP4L activity. In summary, ORP family members promote PI(4,5)P 2 signaling [37•,49]; they also restrict PI(4,5)P 2 signaling [39•,42]; they have been shown to be lipid transfer proteins [31][32][33][34][35]; they have also been described as lipid transmission proteins [40,41]; they are necessary for cancer cell proliferation, survival, and migration [46,48,49]; they have also been described as tumor cell suppressors [40].…”

“…ORP4L phosphorylation enhances cholesterol binding, possibly at the expense of PI(4,5)P 2 binding [49], providing a potential regulatory mechanism for ORP4L activity. In summary, ORP family members promote PI(4,5)P 2 signaling [37•,49]; they also restrict PI(4,5)P 2 signaling [39•,42]; they have been shown to be lipid transfer proteins [31][32][33][34][35]; they have also been described as lipid transmission proteins [40,41]; they are necessary for cancer cell proliferation, survival, and migration [46,48,49]; they have also been described as tumor cell suppressors [40]. Following IP 3 receptor-mediated ER Ca 2+ release, the ER Ca 2+ sensor STIM1 oligomerizes and translocates to ER-PM contacts where it activates store-operated Ca 2+ entry (SOCE) via the Orai1 Ca 2+ channel in the PM ( Figure 1B) [14][15][16].…”