Maria Jolanta Redowicz

Regulation of nonmuscle myosins by heavy chain phosphorylation.

Functional activities of many nonmuscle myosin isoforms are (or are postulated to be) regulated by heavy chain phosphorylation. Depending on the myosin isoform, the serine or threonine residues located within the head (myosin I or myosin VI) or within the C-terminal tail domains (myosin II or myosin V) can be phosphorylated by more or less specific endogenous kinases. In some isoforms phosphorylation can occur both in the head and tail domains, as it has been found for myosin III. There are also isoforms that can be regulated both by the heavy and regulatory light chain phosphorylation, as for the example myosin II from slide mold Dictyostelium discoideum. The goal of this review was to describe recent findings on regulation of myosin I, myosin II, myosin III, myosin V and myosin VI isoforms by their heavy chain phosphorylation including the short charcteristics of the relevant kinases. The biological aspects of the phosphorylation are also discussed.Functional activities of many nonmuscle myosin isoforms are (or are postulated to be) regulated by heavy chain phosphorylation. Depending on the myosin isoform, the serine or threonine residues located within the head (myosin I or myosin VI) or within the C-terminal tail domains (myosin II or myosin V) can be phosphorylated by more or less specific endogenous kinases. In some isoforms phosphorylation can occur both in the head and tail domains, as it has been found for myosin III. There are also isoforms that can be regulated both by the heavy and regulatory light chain phosphorylation, as for the example myosin II from slide mold Dictyostelium discoideum. The goal of this review was to describe recent findings on regulation of myosin I, myosin II, myosin III, myosin V and myosin VI isoforms by their heavy chain phosphorylation including the short charcteristics of the relevant kinases. The biological aspects of the phosphorylation are also discussed.

Emerging Roles of Ruk/CIN85 in Vesicle‐Mediated Transport, Adhesion, Migration and Malignancy

Ruk/CIN85 is an adaptor protein. Similar to many other proteins of this type, Ruk/CIN85 is known to take part in multiple cellular processes including signal transduction, vesicle‐mediated transport, cytoskeleton remodelling, programmed cell death and viral infection. Recent studies have also revealed the potential importance of Ruk/CIN85 in cancer cell invasiveness. In this review we summarize the various roles of this protein as well as the potential contribution of Ruk/CIN85 to malignancy and the invasiveness of cancer cells. In the last section of the paper we also speculate on the utility of Ruk/CIN85 as a target for novel anti‐cancer therapies.

Myosins and deafness.

The discovery in the past few years of a huge diversity within the myosin superfamily has been coupled with an understanding of the role of these motor proteins in various cellular functions. Extensive studies have revealed that myosin isoforms are not only involved in muscle contraction but also in crucial functions of many specialized mammalian cells such as melanocytes, kidney and intestinal brush border microvilli, nerve growth cones or inner ear hair cells. A search for genes involved in the pathology of human genetic deafness resulted in identification of three novel myosins: myosin VI, myosin VIIA and, very recently, myosin XV. The structure, tissue and cellular distribution of these myosin isoforms, as well as mutations detected within their genes that have been found to affect the hearing process, are described in this review.

Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

BackgroundRuk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins.ResultsIn the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division.ConclusionObtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division.

Myosin VI in PC12 cells plays important roles in cell migration and proliferation but not in catecholamine secretion

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.

A novel mutation in the DNM2 gene impairs dynamin 2 localization in skeletal muscle of a patient with late onset centronuclear myopathy

Centronuclear myopathies constitute a group of heterogeneous congenital myopathies characterized by the presence of abnormal, centrally located nuclei within muscle fibers. Centronuclear myopathies can be caused by mutations of several different genes, including DNM2, encoding dynamin 2 (DNM2) a large GTPase involved in membrane trafficking and endocytosis. We report a 52-year-old female with slowly progressive muscle weakness, and a family history of the disease. Clinical, morphological, biochemical and genetic analyses of the proband and her family members were performed, including analyses of the probands muscle biopsy. A novel D614N mutation, located in the C-terminal region pleckstrin-homology (PH) domain of DNM2 was identified in the proband and four family members, who exhibited similar symptoms. The mutation was associated with profound changes in the localization of DNM2 in muscle fibers without significant changes in protein expression. Mutated DNM2 and proteins involved in the membrane trafficking or membrane compartments maintenance were dislocalized within the myofiber, and concentrated at centrally located nuclei. This novel causative mutation (D614N) within the DNM2 gene in a large Polish centronuclear myopathy family with a late age of overt clinical manifestation caused profound changes in DNM2 localization and impaired proper organization of myofibers, and skeletal muscle functioning.

CacyBP/SIP as a novel modulator of the thin filament.

The CacyBP/SIP protein interacts with several targets, including actin. Since the majority of actin filaments are associated with tropomyosin, in this work we characterized binding of CacyBP/SIP to the actin-tropomyosin complex and examined the effects of CacyBP/SIP on actin filament functions. By using reconstituted filaments composed of actin and AEDANS-labeled tropomyosin, we observed that binding of CacyBP/SIP caused an increase in tropomyosin fluorescence intensity indicating the occurrence of conformational changes within the filament. We also found that CacyBP/SIP bound directly to tropomyosin and that these proteins did not compete with each other for binding to actin. Electron microscopy showed that in the absence of tropomyosin CacyBP/SIP destabilized actin filaments, but tropomyosin reversed this effect. Actin-activated myosin S1 ATPase activity assays, performed using a colorimetric method, indicated that CacyBP/SIP reduced ATPase activity and that the presence of tropomyosin enhanced this inhibitory effect. Thus, our results suggest that CacyBP/SIP, through its interaction with both actin and tropomyosin, regulates the organization and functional properties of the thin filament.

Involvement of unconventional myosin VI in myoblast function and myotube formation

The important role of unconventional myosin VI (MVI) in skeletal and cardiac muscle has been recently postulated (Karolczak et al. in Histochem Cell Biol 139:873–885, 2013). Here, we addressed for the first time a role for this unique myosin motor in myogenic cells as well as during their differentiation into myotubes. During myoblast differentiation, the isoform expression pattern of MVI and its subcellular localization underwent changes. In undifferentiated myoblasts, MVI-stained puncti were seen throughout the cytoplasm and were in close proximity to actin filaments, Golgi apparatus, vinculin-, and talin-rich focal adhesion as well as endoplasmic reticulum. Colocalization of MVI with endoplasmic reticulum was enhanced during myotube formation, and differentiation-dependent association was also seen in sarcoplasmic reticulum of neonatal rat cardiomyocytes (NRCs). Moreover, we observed enrichment of MVI in myotube regions containing acetylcholine receptor-rich clusters, suggesting its involvement in the organization of the muscle postsynaptic machinery. Overexpression of the H246R MVI mutant (associated with hypertrophic cardiomyopathy) in myoblasts and NRCs caused the formation of abnormally large intracellular vesicles. MVI knockdown caused changes in myoblast morphology and inhibition of their migration. On the subcellular level, MVI-depleted myoblasts exhibited aberrations in the organization of actin cytoskeleton and adhesive structures as well as in integrity of Golgi apparatus and endoplasmic reticulum. Also, MVI depletion or overexpression of H246R mutant caused the formation of significantly wider or aberrant myotubes, respectively, indicative of involvement of MVI in myoblast differentiation. The presented results suggest an important role for MVI in myogenic cells and possibly in myoblast differentiation.

Arginine deprivation induces endoplasmic reticulum stress in human solid cancer cells.

Deprivation for the single amino acid arginine is a rapidly developing metabolic anticancer therapy, which allows growth control in a number of highly malignant tumors. Here we report that one of the responses of human solid cancer cells to arginine starvation is the induction of prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Systematic study of two colorectal carcinoma HCT-116 and HT29, glioblastoma U251 MG and ovarian carcinoma SKOV3 cell lines revealed, however, that the ER stress triggered by the absence of arginine does not result in massive apoptosis despite a profound upregulation of the proapoptotic gene CHOP. Instead, Akt- and MAPK-dependent pathways were activated which may counteract proapoptotic signaling. Treatment with DMSO as a disaggregating agent or with cycloheximide to block protein synthesis reduced ER stress evoked by arginine deprivation. On the other hand, ER stress and apoptosis induction in arginine-starved cells could be critically augmented by the arginine analog of plant origin canavanine, but not by the classic ER stress inducer tunicamycin. Our data suggest that canavanine treatment applied under the lack of arginine may enhance the efficacy of arginine deprivation-based anticancer therapy.