Biomedical subjects

A Elzagallaai

Publications and source records attributed to A Elzagallaai.

5 recordsLinked to original sources

PubMed2001

Myristoylated alanine-rich C kinase substrate phosphorylation is involved in thrombin-induced serotonin release from platelets.

Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

Blood Platelets↗

PubMed2001

An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization-induced cortical F-actin disassembly and exocytosis.

Chromaffin cell secretion requires cortical F-actin disassembly and it has been suggested that scinderin, a Ca2+ dependent F-actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 microM scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F-actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization-evoked cortical F-actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin-permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation-induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (B(max)) secretory response to Ca2+ without modifying the affinity (K(m)) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine ss-hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle-plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.

Actins↗

PubMed2000

Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKS-derived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation.

Previous experiments suggest that actin disassembly, perhaps at a specific site, is required for platelet secretion. Platelet stimulation by phorbol 12-myristate 13-acetate (PMA) induced pleckstrin phosphorylation, platelet aggregation, and secretion. Inhibition of protein kinase C (PKC) is accompanied by inhibition of pleckstrin phosphorylation and serotonin secretion. Here, we demonstrate the presence of myristoylated alanine-rich C kinase substrate (MARCKS), another PKC substrate, in platelets and its phosphorylation during PMA stimulation. MARCKS is known to bind actin and to cross-link actin filaments; the latter is inhibited by PKC-induced MARCKS phosphorylation. MARCKS phosphorylation and serotonin release from permeabilized platelets have the same time course and were blocked by a peptide (MPSD) with the amino acid sequence corresponding to the phosphorylation site domain of MARCKS. Pleckstrin and myosin light chain phosphorylation was not modified. A peptide (Ala-MPSD) in which the four serine residues of MPSD were substituted by alanines was ineffective. These results provide the first evidence that MARCKS may play a role in platelet secretion. Moreover, pleckstrin phosphorylation has a different time course than that of MARCKS or serotonin release and was not modified when MARCKS phosphorylation and serotonin release were inhibited, suggesting that pleckstrin is either not directly involved in secretion or that it might only be involved upstream in the cascade of events leading to exocytosis.

Amino Acid Sequence↗

PubMed2000

Two pathways control chromaffin cell cortical F-actin dynamics during exocytosis.

Neurosecretory cells including chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane. We have proposed that the F-actin network acts as a barrier to the secretory vesicles blocking their access to exocytotic sites at the plasma membrane. Disassembly of cortical F-actin in chromaffin cells in response to stimulation is thought to allow the free movement of secretory vesicles to exocytotic sites. Moreover, experiments by us using morphometric analysis of resting and stimulated chromaffin cells together with membrane capacitance measurements have shown that cortical F-actin controls the traffic of vesicles from the vesicle reserve compartment to the release-ready vesicle compartment. The dynamics of the cortical F-actin is controlled by two pathways: A) stimulation-induced Ca(2+) entry and scinderin activation; and B) protein kinase C (PKC) activation and MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation. When chromaffin cells are stimulated through nicotinic receptors, cortical F-actin disassembly is mainly through the intervention of pathway A, since in the presence of PKC inhibitors, F-actin disassembly in response to cholinergic stimulation is only blocked by 20%. Pathway A involves the activation of scinderin by Ca(2+) with a consequent F-actin severing. Pathway B is fully activated by phorbol esters and in this case PKC blockers inhibit by 100% the disruption of cortical F-actin. This pathway operates through MARCKS. A peptide with amino acid sequence corresponding to the phosphorylation site domain of MARCKS, which also corresponds to its actin binding site, blocks PMA potentiation of Ca(2+)-induced catecholamine release. The results suggest that under physiological conditions (i.e., nicotinic receptor stimulation) pathway A is the principal mechanism for the control of cortical F-actin dynamic changes.

Actins↗

PubMed1998

Localization by segmental deletion analysis and functional characterization of a third actin-binding site in domain 5 of scinderin.

Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin-evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle exocytosis (Vitale, M. L., Rodríguez Del Castillo, A., Tchakarov, L., and Trifaró, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding site at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc502-715) containing this site binds to actin-DNase-I-Sepharose 4B beads, co-sediments with actin and is able to nucleate actin assembly. Recombinant ScL5-6, a fusion protein devoid of the actin-binding site (Sc519-715), did not exhibit these properties. Moreover, Sc-ABP3, a peptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP3 also prevented the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release of serotonin from permeabilized platelets. On the other hand, ScL5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1-4,6, a construct devoid of domain 5, was able to sever but unable to nucleate actin, indicating that an actin nucleation site of scinderin was in domain 5. The results suggest that scinderin, in addition to binding actin on sites present in domains 1 and 2, must bind actin on a third site in domain 5 to sever and nucleate actin effectively.

Actins↗