Characterization of muscle filamin isoforms suggests a possible role of gamma-filamin/ABP-L in sarcomeric Z-disc formation

Filamin, also called actin binding protein-280, is a dimeric protein that cross-links actin filaments in the cortical cytoplasm. In addition to this ubiquitously expressed isoform (FLN1), a second isoform (ABP-L/gamma-filamin) was recently identified that is highly expressed in mammalian striated muscles. A monoclonal antibody was developed, that enabled us to identify filamin as a Z-disc protein in mammalian striated muscles by immunocytochemistry and immunoelectron microscopy. In addition, filamin was identified as a component of intercalated discs in mammalian cardiac muscle and of myotendinous junctions in skeletal muscle. Northern and Western blots showed that both, ABP-L/gamma-filamin mRNA and protein, are absent from proliferating cultured human skeletal muscle cells. This muscle specific filamin isoform is, however, up-regulated immediately after the induction of differentiation. In cultured myotubes, ABP-L/gamma-filamin localises in Z-discs already at the first stages of Z-disc formation, suggesting that ABP-L/gamma-filamin might play a role in Z-disc assembly.     

Calpain 1-titin interactions concentrate calpain 1 in the Z-band edges and in the N2-line region within the skeletal myofibril

Calpain 1, a ubiquitous calcium-dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci79, 2097-2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z-band and at the midpoint of the I-band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I-band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N-terminal Z8-I5 region and the N1-line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl(2) and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z-band edge and the N1-line as well as at the N2-line level, two sarcomeric regions where early postmortem proteolysis is detected.     

Evidence that an isoform of calpain-10 is a regulator of exocytosis in pancreatic beta-cells

Calpain-10 (CAPN10) is the first type 2 diabetes susceptibility gene to be identified through a genome scan, with polymorphisms being associated with altered CAPN10 expression. Functional data have been hitherto elusive, but we report here a corresponding increase between CAPN10 expression level and regulated insulin secretion. Pancreatic beta-cell secretory granule exocytosis is mediated by the soluble N-ethylmaleimide-sensitive fusion protein attachment receptor protein complex of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin 1, and vesicle-associated membrane protein 2. We report, for the first time, direct binding of a calpain-10 isoform with members of this complex. Furthermore, SNAP-25 undergoes a Ca2+-dependent partial proteolysis during exocytosis, with calpain protease inhibitor similarly suppressing both insulin secretion and SNAP-25 proteolysis. Based upon these findings, we postulate that an isoform of calpain-10 is a Ca2+-sensor that functions to trigger exocytosis in pancreatic beta-cells.     

Activation of protein kinase C zeta induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle

Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKC zeta in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKC zeta to associate specifically with the GLUT4 compartments and that PKC zeta together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKC zeta and GLUT4 recycled independently of one another. To further establish the importance of PKC zeta in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKC zeta (DNPKC zeta) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKC zeta was associated with a marked increase in the activity of this isoform. The overexpressed, active PKC zeta coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKC zeta caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKC zeta induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKC zeta disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKC zeta regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.     

Requirement of protein kinase C micro activation and calpain-mediated proteolysis for arachidonic acid-stimulated adhesion of MDA-MB-435 human mammary carcinoma cells to collagen type IV

Arachidonic acid (AA) stimulation of adhesion of human metastatic breast carcinoma cells to collagen type IV depends on the protein kinase C (PKC) pathway(s) and is associated with the translocation of PKC mu from the cytoplasm to the membrane. In the present study, we have further explored the role of PKC mu in AA-stimulated adhesion. PKC mu activation site serines 738/742 and autophosphorylation site serine 910 are rapidly phosphorylated, and in vitro PKC mu kinase activity is enhanced in response to AA treatment. Inhibition of PKC mu activation blocks AA-stimulated adhesion. A phosphorylated, truncated species of PKC mu was detected in AA-treated cells. This 77-kDa protein contains the kinase domain but lacks a significant portion of the regulatory domains. Inhibition of calpain protease activity blocks generation of the truncated protein, promotes accumulation of the activated, full-length protein in the membrane, and blocks the AA-mediated increase in adhesion. p38 MAPK activity is also required for AA-stimulated adhesion. Activation of PKC mu and p38 are independent events. However, inhibition of p38 activity reduces calpain-mediated proteolysis of PKC mu and in vivo calpain activity, suggesting a role for p38 in regulation of calpain activity and a point for cross-talk between the PKC and MAPK pathways. These results support the hypothesis that AA stimulates activation of PKC mu, which is cleaved by calpain at the cell membrane. The resulting truncated kinase, as well as the full-length kinase, may be required for increased cell adhesion to collagen type IV. Additionally, these studies present the first evidence for calpain cleavage of a non-structural protein leading to the promotion of tumor cell adhesion.     

Phosphoinositide binding to the substrate regulates susceptibility to proteolysis by calpain

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein alpha-actinin to proteolysis by calpains 1 and 2. At first, alpha-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) binding to alpha-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave alpha-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P(3) binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of alpha-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P(3), alpha-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of alpha-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P(3)-mediated calpain proteolysis of alpha-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P(3) binding is a critical determinant for alpha-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.     

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.     

H1 histone stimulates limited proteolysis of protein kinase C subspecies by calpain II.

Limited proteolysis of protein kinase C (PKC) subspecies with Ca2(+)-dependent neutral protease II (calpain II) was remarkably stimulated by basic polypeptides, such as H1 histone and poly-L-lysine. This stimulatory effect was observed for proteolysis of the active form of PKC, which was associated with phospholipid and diacylglycerol. The inactive form of PKC was far less susceptible to proteolysis, both in the presence and absence of the basic polypeptides. The basic polypeptides did not appear to interact with calpain II, but made the PKC molecule more susceptible to proteolysis. The relative rates of cleavage of type I (gamma), II (beta), and III (alpha) PKC were 2:2:1. The available evidence suggests that, like calpain I, calpain II may also contribute to the down-regulation or depletion of PKC.     

MNB/DYRK1A phosphorylation regulates the interactions of synaptojanin 1 with endocytic accessory proteins

MNB/DYRK1A is a proline-directed serine/threonine kinase implicated in Down syndrome (DS). In an earlier screening, two proteins from adult rat brain, one 100kDa and the other 140 kDa, were found to be prominently phosphorylated by the kinase. The 100-kDa protein was previously characterized as an isoform of dynamin 1. In this study, we identified the 140-kDa protein as synaptojanin 1 (SJ1). MNB/DYRK1A phosphorylates SJ1 at multiple sites and produces complex behaviors in binding to amphiphysin 1 and intersectin 1 (ITSN1). However, the phosphorylation has little effect on the phosphatidylinositol phosphatase activity of SJ1. These results suggest that MNB/DYRK1A is involved in regulating the recruitment activity but not the phosphatase activity of SJ1. Our findings may be especially important in the etiology of DS because MNB/DYRK1A, SJ1, and ITSN1 are all located at or near the region of human chromosome 21, which is postulated to be involved in the disease.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

Calcium-Dependent Cleavage of Striatal Enriched Tyrosine Phosphatase (STEP)

Striatal enriched phosphatase (STEP) is a family of protein tyrosine phosphatases enriched within the CNS. A member of this family, STEP61, is a membrane-associated protein located in postsynaptic densities of striatal neurons. In this study, we demonstrate that STEP61, is cleaved into smaller isoforms. To clarify the mechanism of cleavage, STEP61 was transiently expressed in NT2/D1 neuronal precursor cells. Exposure of transfected cells to the calcium ionophore, A23187, or to thapsigargin resulted in the rapid cleavage of STEP61. Pretreatment with the calpain inhibitor, calpeptin, or EGTA prevented proteolysis. One of the cleavage products has a relative molecular mass of 33 kDa (STEP33). A protein with the identical mobility is detected following calpain treatment of STEP61 fusion protein or postsynaptic densities purified from rat striatum. Exposure of primary neuronal cultures to glutamate also led to a significant increase in the concentration of a low molecular weight form of STEP. Taken together, these results suggest that in response to a rapid influx of calcium, STEP61, is proteolytically cleaved by calpain, leading to the release of a smaller isoform. This model may explain the rapid appearance of STEP33 in response to transient hypoxia-ischemia in the brain as cells attempt to counter the increase in tyrosine phosphorylation levels following neuronal insults.     

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.     

Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function

Individual isoforms of the protein kinase C (PKC) family of kinases may have assumed distinct responsibilities for the control of complex and diverse cellular functions. In this study, we show that an isoform specific interaction between PKC epsilon and filamentous actin may serve as a necessary prelude to the enhancement of glutamate exocytosis from nerve terminals. Using a combination of cosedimentation, overlay, and direct binding assays, we demonstrate that filamentous actin is a principal anchoring protein for PKC epsilon within intact nerve endings. The unusual stability and direct nature of this physical interaction indicate that actin filaments represent a new class of PKC- binding protein. The binding of PKC epsilon to actin required that the kinase be activated, presumably to expose a cryptic binding site that we have identified and shown to be located between the first and second cysteine-rich regions within the regulatory domain of only this individual isoform of PKC. Arachidonic acid (AA) synergistically interacted with diacylglycerol to stimulate actin binding to PKC epsilon. Once established, this protein-protein interaction securely anchored PKC epsilon to the cytoskeletal matrix while also serving as a chaperone that maintained the kinase in a catalytically active conformation. Thus, actin appears to be a bifunctional anchoring protein that is specific for the PKC epsilon isoform. The assembly of this isoform-specific signaling complex appears to play a primary role in the PKC-dependent facilitation of glutamate exocytosis.     

Indications for a novel muscular dystrophy pathway. gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin

gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs. A minor fraction of the protein shows subsarcolemmal localization. Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20. Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon. Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs. The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin. The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin. Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease.     

Protein kinase C inhibits binding of diacylglycerol kinase-zeta to the retinoblastoma protein

We previously showed that the retinoblastoma protein (pRB), a key regulator of G1 to S-phase transition of the cell cycle, binds to and stimulates diacylglycerol kinase-zeta (DGKzeta) to phosphorylate the lipid second messenger diacylglycerol into phosphatidic acid. pRB binds to the MARCKS phosphorylation-site domain of DGKzeta that can be phosphorylated by protein kinase C (PKC). Here, we report that activation of PKC by phorbol ester inhibits DGKzeta binding to pRB. Ro 31-8220, a specific inhibitor of PKC, alleviated this inhibition of binding. Mimicking of PKC phosphorylation of serine residues (by S/D but not S/N mutations) within the DGKzeta-MARCKS phosphorylation-site domain also prevented DGKzeta binding to pRB, suggesting that PKC phosphorylation of these residues negatively regulates the interaction between DGKzeta and pRB. In PKC overexpression studies, it appeared that activation of particularly the (wild-type) PKCalpha isoform inhibits DGKzeta binding to pRB, whereas dominant-negative PKCalpha neutralized this inhibition. PKCalpha activation thus prevents DGKzeta regulation by pRB, which may have implications for nuclear diacylglycerol and phosphatidic acid levels during the cell cycle.     

Isoform specific function of calpain 2 in regulating membrane protrusion

Previous studies have demonstrated a role for calpains in cell migration through their capacity to regulate focal adhesion dynamics and rear retraction. In this study, we provide evidence that calpains also modulate membrane protrusion activity in fibroblasts. We find that an immortalized Capn4(-/-) fibroblast line displays an altered morphology, characterized by numerous thin membrane projections and increased transient membrane activity. Furthermore, we show that protrusion kinetics of lamellipodia at the leading edge are improperly regulated in Capn4(-/-) cells, leading to impaired net forward lamellipodial extension. To address the isoform specific functions of calpain 1 and calpain 2 during cell protrusion, we stably introduced small interfering RNAs (siRNAs) targeting each isoform into a fibroblast cell line. Despite a loss in calpain 1 activity, calpain 1 knockdown cells show normal morphology and membrane protrusion dynamics. However, cells in which calpain 2 is knocked down are characterized by a protrusive morphology, increased transient membrane activity and altered protrusion kinetics, similar to the Capn4(-/-) fibroblasts. Additionally, we find that calpain 2, but not calpain 1, is required for proteolysis of the cytoskeletal and focal adhesion proteins FAK, paxillin, spectrin, and talin. Together, our findings support a novel role for calpain 2 in limiting membrane protrusions and in regulating lamellipodial dynamics at the leading edge of migrating cells.     

Presence of Phosphatidylserine Synthesizing Enzymes in Triton Insoluble Floating Fractions from Cerebrocortical Plasma Membranes

Mammals synthesize phosphatidylserine (PS), a binding PKC molecule, by exchanging the nitrogen base of phosphatidylethanolamine or phosphatidylcholine with free serine. Serine base exchange enzyme (SBEE) was found in Triton insoluble floating fractions (TIFFs) from rat cerebellum which contained PKC. Consequently, SBEE might modulate PS levels in the PKC binding area (Buratta et al., J Neurochem 103:942–951, 2007). In the present study, we determined whether SBEE and PKC were localised in rat cerebral cortex TIFFs (cx-TIFFs) and in rat cerebrocortical plasma membrane-TIFFs (PM-TIFFs) which are more directly involved in signal transduction than intracellular membranes. Cx-and PM-TIFFs expressed SBEE activity and contained PKC. SBEE used ethanolamine as free exchanging base which may modulate PS level in the PKC binding area, transforming PS into PE and vice versa. The slight decrease in [¹⁴C]serine incorporation in the presence of choline indicated the existence of a SBEE isoform which may play a peculiar role in this brain area.