Maria A. Brehm

Human inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) is a nucleocytoplasmic shuttling protein specifically enriched at cortical actin filaments and at invaginations of the nuclear envelope.

Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P3 phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal (134LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB (129RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104–165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P3-mediated Ca2+ signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca2+ signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P3 but may also be involved in modulating nuclear Ca2+ signals generated from these nuclear envelope invaginations.

Tumour cells can employ extracellular Ins(1,2,3,4,5,6)P(6) and multiple inositol-polyphosphate phosphatase 1 (MINPP1) dephosphorylation to improve their proliferation.

InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 μM. We noticed that InsP6 in culture medium at a concentration of ≤50 μM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 μM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.

Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)

Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41-49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108-170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B-EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)-Rattus norvegicus IP3K (aa 108-170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.

Force-Sensitive Autoinhibition of the von Willebrand Factor Is Mediated by Interdomain Interactions

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis.

Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation.

Abstract Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], as well as mTOR-raptor.

The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei.

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

Mutual A domain interactions in the force sensing protein von Willebrand factor

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

A non-catalytic role for inositol 1,3,4,5,6-pentakisphosphate 2-kinase in the synthesis of ribosomal RNA.

Summary Fundamental to the life and destiny of every cell is the regulation of protein synthesis through ribosome biogenesis, which begins in the nucleolus with the production of ribosomal RNA (rRNA). Nucleolar organization is a highly dynamic and tightly regulated process; the structural factors that direct nucleolar assembly and disassembly are just as important in controlling rRNA synthesis as are the catalytic activities that synthesize rRNA. Here, we report that a signaling enzyme, inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5K) is also a structural component in the nucleolus. We demonstrate that IP5K has functionally significant interactions with three proteins that regulate rRNA synthesis: protein kinase CK2, TCOF1 and upstream-binding-factor (UBF). Through molecular modeling and mutagenic studies, we identified an Arg-Lys-Lys tripeptide located on the surface of IP5K that mediates its association with UBF. Nucleolar IP5K spatial dynamics were sensitive to experimental procedures (serum starvation or addition of actinomycin D) that inhibited rRNA production. We show that IP5K makes stoichiometrically sensitive contributions to the architecture of the nucleoli in intact cells, thereby influencing the degree of rRNA synthesis. Our study adds significantly to the biological significance of IP5K; previously, it was the kinase activity of this protein that had attracted attention. Our demonstration that IP5K ‘moonlights’ as a molecular scaffold offers an unexpected new example of how the biological sophistication of higher organisms can arise from gene products acquiring multiple functions, rather than by an increase in gene number.

Biophysical approaches promote advances in the understanding of von Willebrand factor processing and function.

The large multimeric plasma glycoprotein von Willebrand factor (VWF) is essential for primary hemostasis by recruiting platelets to sites of vascular injury. VWF multimers respond to elevated hydrodynamic forces by elongation, thereby increasing their adhesiveness to platelets. Thus, the activation of VWF is force-induced, as is its inactivation. Due to these attributes, VWF is a highly interesting system from a biophysical point of view, and is well suited for investigation using biophysical approaches. Here, we give an overview on recent studies that predominantly employed biophysical methods to gain novel insights into multiple aspects of VWF: Electron microscopy was used to shed light on the domain structure of VWF and the mechanism of VWF secretion. High-resolution stochastic optical reconstruction microscopy, atomic force microscopy (AFM), microscale thermophoresis and fluorescence correlation spectroscopy allowed identification of protein disulfide isomerase isoform A1 as the VWF dimerizing enzyme and, together with molecular dynamics simulations, postulation of the dimerization mechanism. Advanced mass spectrometry led to detailed identification of the glycan structures carried by VWF. Microfluidics was used to illustrate the interplay of force and VWF function. Results from optical tweezers measurements explained mechanisms of the force-dependent functions of VWFs domains A1 and A2 and, together with thermodynamic approaches, increased our understanding of mutation-induced dysfunctions of platelet-binding. AFM-based force measurements and AFM imaging enabled exploration of intermonomer interactions and their dependence on pH and divalent cations. These advances would not have been possible by the use of biochemical methods alone and show the benefit of interdisciplinary research approaches.

Efficacious inhibition of Importin α/β-mediated nuclear import of human inositol phosphate multikinase.

Human inositol phosphate multikinase (IPMK) is a nucleocytoplasmic shuttling protein involved in multiple signal transduction pathways located both in the nucleus and in the cytoplasm. To efficaciously inhibit the conventional nuclear import of IPMK, we first examined the effect of different inhibitors and cellular stressors on nuclear import of enhanced green fluorescent protein monomer and octamer, both fused with a monopartite nuclear localization signal (NLS), in HeLa and H1299 cells. Most efficacious inhibition of conventional nuclear protein import was observed when using Importazole and hydrogen peroxide. Therefore, these substances were then applied to examine nuclear import mechanisms of IPMK. Thereby, we demonstrated that nuclear accumulation of IPMK is significantly lessened, but not abrogated by inhibition of conventional protein import. This indicates that IPMK is imported into the nucleus by both conventional and non-conventional pathways. Furthermore, intracellular distribution of an IPMK mutant with inactivated NLS is unaffected by inhibition of conventional protein import. Obviously, the conventional import of IPMK is entirely mediated by interaction of the Importin α/β heterodimer with IPMKs sole NLS motif (R(320)HRKIYTKKHH). Future research should focus on the hitherto unknown non-conventional import of IPMK and the potential impact of its dysregulation on IPMK signaling pathways regulating cellular growth and proliferation.