scholarly journals InsP3-mediated intracellular calcium signalling is altered by expression of synaptojanin-1

2004 ◽  
Vol 382 (2) ◽  
pp. 687-694 ◽  
Author(s):  
Friedrich W. JOHENNING ◽  
Markus R. WENK ◽  
Per UHLÉN ◽  
Brenda DeGRAY ◽  
Eunkyung LEE ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Membrane Trafficking ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Calcium Signalling ◽  
Physiological Role ◽  
Actin Dynamics ◽  
Phosphatase Domain ◽  
Phosphoinositide Phosphatase ◽  
Synaptojanin 1

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a phosphoinositide phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals.

scholarly journals The cytoplasmic domain of P-selectin is phosphorylated on serine and threonine residues

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1758-1766 ◽  
Author(s):  
T Fujimoto ◽  
RP McEver
Keyword(s):  
Endothelial Cells ◽  
Membrane Trafficking ◽  
Cell Activation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Ovary Cells ◽  
Phosphatase Inhibitors ◽  
Protein Kinase C Inhibitors ◽  
Activated Platelets

Abstract P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.

scholarly journals Absence of Sac2/INPP5F enhances the phenotype of a Parkinson’s disease mutation of synaptojanin 1

2020 ◽  
Vol 117 (22) ◽  
pp. 12428-12434 ◽  
Author(s):  
Mian Cao ◽  
Daehun Park ◽  
Yumei Wu ◽  
Pietro De Camilli
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Gene Encoding ◽  
Phosphatase Domain ◽  
Inositol Phosphatase ◽  
Phosphoinositide Phosphatase ◽  
Abnormal Accumulation ◽  
Synaptojanin 1

Numerous genes whose mutations cause, or increase the risk of, Parkinson’s disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a “synthetic” effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

scholarly journals Similarity between physicochemical properties of recombinant rat prorenin and native inactive renin

1991 ◽  
Vol 275 (3) ◽  
pp. 727-731 ◽  
Author(s):  
M Hosoi ◽  
S Kim ◽  
T Yamauchi ◽  
T Watanabe ◽  
K Murakami ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Physiological Role ◽  
Immunoblot Analysis ◽  
Physicochemical Characteristics ◽  
Rat Plasma ◽  
Cdna Sequences ◽  
Sepharose Column ◽  
C Terminus ◽  
Inactive Renin

Rat prorenin was synthesized by Chinese-hamster ovary cells transfected with an expression vector containing rat preprorenin cDNA sequences, then purified by concanavalin A-Sepharose chromatography and h.p.l.c. on G3000SW. The molecular mass of purified prorenin was 46,000 Da, as determined by h.p.l.c. on G3000SW. Immunoblot analysis indicated that recombinant prorenin cross-reacted with anti-(mature renin) antibody and two kinds of antibodies recognizing the N-terminus and C-terminus of the prosegment of rat prorenin. Recombinant prorenin was bound to a Cibacron Blue-Sepharose column and eluted with 1.4 M-NaCl, but was not retained by an octapeptide renin inhibitor (H-77)-Sepharose column. Trypsin activation of prorenin increased the renin activity 110-fold, caused binding to an H-77-Sepharose column and nullified the reactivity to the above two kinds of anti-prosegment antibodies, findings indicating that the activation of prorenin with trypsin is due to the cleavage of the prosegment. Rat plasma inactive renin, partially purified by h.p.l.c. on G3000SW, had much the same physicochemical characteristics as the recombinant prorenin. These results provide evidence that rat plasma inactive renin is prorenin. Recombinant prorenin is a useful material for examining the physiological role of circulating prorenin.

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