Dephosphorylation of beta2-syntrophin and Ca2+/micro-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Abstract

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic b-cells. Binding of its cytoplasmic tail to b2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca 2+ -dependent proteolysis of ICA512. In vitro m-calpain cleaves ICA512 between a putative PEST domain and the b2-syntrophin binding site, whereas binding of ICA512 to b2-syntrophin protects the former from cleavage. b2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-b2-syntrophin and stimulation of insulin secretion induces the Ca 2+ -dependent, okadaic acid-sensitive dephosphorylation of b2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from b2-syntrophin±utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by m-calpain. Keywords: actin/diabetes/IA-2/secretory granule/tyrosine phosphatase IntroductionThe exocytosis of neurosecretory granules is associated with a focal and transient reorganization of the cortical actin cytoskeleton. Actin may control secretion in a variety of ways. For instance, evidence that depolymerization of actin micro®laments increases stimulated secretion Trifaro, 1990) led to the suggestion that the actin web acts as a barrier that prevents granules from approaching sites of exocytosis (Sontag et al., 1988;Vitale et al., 1991;Nakata and Hirokawa, 1992). The reorganized actin ®laments can also directly support the movement of granules across the subcortical region (Lang et al., 2000). Actin bundles and ®lamentous proteins, in addition, may tether secretory granules to each other and to the plasma membrane, thereby restraining vesicle mobility (Senda et al., 1988(Senda et al., , 1989Lang et al., 2000). Thus, the dynamic relationship of secretory granules with the actin network is likely to account, at least in part, for the existence of different pools of secretory granules, which differ in respect to their readiness to undergo exocytosis upon stimulation of secretion (Trifaro, 1990;Klenchin and Martin, 2000). The identity of the molecule(s) that mediates the interaction between secretory granules and the actin cortex, however, is still unclear.Recent studies from our laboratory have pointed to islet cell autoantigen (ICA) 512, also known as IA-2, as a potential link between secretory granules and the actin cytoskeleton in insulin-secreting cells (b-cells) of the pancreatic islets. ICA512 is an atypical member of the receptor p...

Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

“…The mouse monoclonal antibodies (mAb) were an IgG2a anti-rat CgB (CIRO) from our laboratory [28]; an anti-insulin (Sigma-Aldrich, Milan, Italy) and an anti-human CgB (67-C7-2), gift of W. B. Huttner (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany); an anti-trans-Golgi network (TGN) marker (TGN38) and an anti-GM130 (Golgi complex marker; BD Biosciences, San Jose, CA, USA); an antilysosomal-associated membrane protein 1 (LAMP1) (LYC16; Calbiochem, Darmstadt, Germany); two antiprotein tyrosine phosphatase-like protein-2 (ICA512/IA-2), mICA512cyto, gift of M. Solimena (Technical University of Dresden, Germany) [29] and 76F from our laboratory [30]. INS-1E lysates and incubation media were immunoblotted to assess antibody specificity (Electronic supplementary material [ESM] Fig.…”

Apolipoprotein A-I Activates Protein Kinase Cα Signaling to Phosphorylate and Stabilize ATP Binding Cassette Transporter A1 for the High Density Lipoprotein Assembly

“…In addition, a PDZ protein, ␤2-syntrophin, also interacts with ABC family proteins ABCA1 (59) and MRP2 (60) or another membrane protein, ICA512 (61). Phosphorylation of either ␤2-syntrophin or the membrane protein may regulate the interaction (60), and dissociation of ICA512 from ␤2-syntrophin accelerates its calpainmediated degradation (61).…”

Calpain-generated natural protein fragments as short-lived substrates of the N-end rule pathway

“…2, #12, and Fig. S1D) enters the nucleus and acts as a transcriptional regulator (30,31). Lys609-Ica512 was shortlived in reticulocyte extract (postpulse t 1/2 of ∼6 min), in contrast to Val609-Ica512, which was completely stable (Fig.…”