Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate.

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.     

Molecular characterization of a glycerophosphoinositol transporter in mammalian cells

The glycerophosphoinositols are ubiquitous phosphoinositide metabolites involved in the control of several cell functions. They exert their actions both intracellularly and by rapidly equilibrating across the plasma membrane when added to cells, implying the existence of a transporter for their membrane permeation. Such a transporter, GIT1, has been cloned in yeast. By PSI-BLAST analysis, we have identified the Glut2 transporter as a human-genome candidate ortholog of GIT1. This was supported directly through the use of inhibitors, siRNAs and competition studies of specific uptake of GroPIns in HeLa cells over-expressing human Glut2. These data identify Glut2 as a GroPIns transporter in mammals, and define a physiologically relevant cell-permeation mechanism.     

Biological activities and metabolism of the lysophosphoinositides and glycerophosphoinositols

The lysophospholipids are integral components of the plasma membrane that have often been considered as side products of the phospholipase A2 (PLA2)-dependent production of arachidonic acid and the deacylation/reacylation processes involved in phospholipid homeostasis. Data indicating roles of these lipid derivatives in hormone responses and cell transformation have now led to a different view, and the understanding of their involvement in the modulation of cell function is building up. Here, we will summarise the current knowledge concerning the biological roles of the lysophosphoinositides and the glycerophosphoinositols (their fully deacylated counterparts) in the framework of their known effects, and those of the other lysophospholipids and glycerophospholipids.     

Glycerophosphoinositol-4-phosphate enhances SDF-1alpha-stimulated T-cell chemotaxis through PTK-dependent activation of Vav

Glycerophosphoinositols (GPIs) are water-soluble phosphoinosite metabolites produced by all cell types, whose levels increase in response to a variety of extracellular stimuli, and are particularly high in Ras-transformed cells. GPIs are released to the extracellular space, wherefrom they can be taken up by other cells through a specific transporter. Exogenous GPIs affect a plethora of cellular functions. Among these compounds the most active is GroPIns4P, which affects cAMP levels and PKA-dependent functions through the inhibition of heterotrimeric Gs proteins. GroPIns4P has also recently been found to promote actin cytoskeleton reorganization by inducing Rho and Rac activation through an as yet unidentified mechanism. Here we have assessed the potential effects of GroPIns4P on T-cells. We found that GroPIns4P enhances CXCR4-dependent chemotaxis. This activity results from the capacity of GroPIns4P to activate the Rho GTPase exchange factor, Vav, through an Lck-dependent pathway which also results in activation of the stress kinases JNK and p38. GroPIns4P was also found to activate with a delayed kinetics the Lck-dependent activation of ZAP-70, Shc and Erk1/2. The activities of GroPIns4P were found to be dependent on its capacity to inhibit cAMP production and PKA activation. Collectively, the data provide the first evidence of a role of glycerophosphoinositols as modulators of T-cell signaling and establish a mechanistic basis for the effects of this phosphoinositide derivative on F-actin dynamics.     

Natural products reveal cancer cell dependence on oxysterol-binding proteins

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.     

Secretion machinery at the cell plasma membrane

Secretion is a fundamental cellular process involving the regulated release of intracellular products from cells. Physiological functions such as neurotransmission, or the release of hormones and digestive enzymes, are all governed by cell secretion. Three critical activities occur at the cell plasma membrane to ensure secretion. Membrane-bound secretory vesicles dock, fuse, and expel their contents to the outside via specialized and permanent plasma membrane structures, called porosomes or fusion pores. In recent years, significant progress has been made in our understanding of these three key cellular activities required for cell secretion. The molecular machinery and mechanism involving them is summarized in this article.     

Circahoralian (Ultradian) metabolic rhythms

This review presents data concerning metabolic rhythms with periods close to one hour (20 to 120 min): their occurrence, biochemical organization, nature, and significance for adaptations and age-related changes of cells and organs. Circahoralian (ultradian) rhythms have been detected for cell mass and size, protein synthesis, enzyme activities, concentration of ATP and hormones, cell respiration, and cytoplasm pH. Rhythms have been observed in bacteria, yeasts, and protozoa, as well as in many cells of metazoans, including mammals, in vivo and in cell cultures. In cell populations, the rhythms are organized by direct cell-cell communication. The biochemical mechanism involves membrane signal factors and cytoplasmic processes resulting in synchronization of individual oscillations to a common rhythm. Phosphorylation of proteins is the key process of coordination of protein synthesis and enzyme activity kinetics. The fractal nature of circahoralian rhythms is discussed as well as the involvement of these rhythms in adaptations of the cells and organs. Senescent decrease in rhythm amplitudes and correspondingly in cell-cell communication has been observed. The possibility of remodeling these changes through the intercellular medium has been predicted and experimentally shown. Perspectives for studies of the organizers and disorganizers of cell-cell communication in the intercellular medium along with appropriate receptors are discussed with special emphasis on aging and pathology. One perspective can be more precise definition of the range of normal biochemical and physiological state with the goal of correction of cellular functions.     

Role of protein kinase C in cellular regulation

Protein kinase C (PKC) consists of a family of closely related enzymes ubiquitously present in animal tissues. These enzymes respond to second messengers, Ca2+, diacylglycerol and arachidonic acid, to express their activities at membrane locations. Numerous hormones, neurotransmitters, growth factors and antigens are believed to transmit their signals by activation of a variety of phospholipases to generate these messengers. The various PKC isozymes, which exhibit distinct biochemical characteristics and unique cellular and subcellular localizations, may be differentially stimulated depending on the duration and strength of these messengers. Activation of PKC has been linked to the regulation of cell surface receptors, ion channels, secretion, gene expression, and neuronal plasticity and toxicity. The mechanisms of action of PKC in the regulation of these cellular functions are not entirely clear. Further study to identify the target substrates relevant to the various cellular functions is essential to define the functional diversity of this enzyme family.     

Seprase: an overview of an important matrix serine protease

Seprase or Fibroblast Activation Protein (FAP) is an integral membrane serine peptidase, which has been shown to have gelatinase activity. Seprase has a dual function in tumour progression. The proteolytic activity of Seprase has been shown to promote cell invasiveness towards the ECM and also to support tumour growth and proliferation. Seprase appears to act as a proteolytically active 170-kDa dimer, consisting of two 97-kDa subunits. It is a member of the group type II integral serine proteases, which includes dipeptidyl peptidase IV (DPPIV/CD26) and related type II transmembrane prolyl serine peptidases, which exert their mechanisms of action on the cell surface. DPPIV and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localisation of these protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors and in the degradation of extracellular matrix components that are essential to the cellular migration and matrix invasion that occur during tumour invasion, metastasis and angiogenesis.     

Multiple Functions of BCL-2 Family Proteins

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.     

Targeted mutagenesis of the angiogenic protein CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-heparan sulfate proteoglycan coreceptor-mediated cellular functions

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin alpha6beta1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha6beta1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support alpha6beta1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha6beta1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished alpha6beta1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alphavbeta3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha6beta1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha6beta1-HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha6beta1-HSPG-specific activities in vivo.     

Roles of nectins in cell adhesion, migration and polarization

Nectins are Ca2+-independent immunoglobulin (Ig)-like cell-cell adhesion molecules, which comprise a family consisting of four members. Nectins have five activities: (1) they show Ca2+-independent cell-cell adhesion activity by homo- and hetero-trans-interactions through their extracellular regions; (2) they bind afadin, an actin filament (F-actin)-binding protein, through their cytoplasmic tails and are connected to the actin cytoskeleton; (3) they induce activation of Cdc42 and Rac small G proteins through their cytoplasmic tails; (4) they bind Par-3, a cell polarity protein, through their cytoplasmic tails; and (5) they heterophilically trans-interact with Necls, nectin-like molecules, through their extracellular regions. Through these activities, nectins regulate a variety of cellular functions, including adhesion, migration, and polarization. Here we describe these activities and functions of nectins.     

JmjC-domain-containing proteins and histone demethylation

Histone methylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity. Enzymes that directly remove methyl marks from histones have recently been identified, revealing a new level of plasticity within this epigenetic modification system. Here we analyse the evolutionary relationship between Jumonji C (JmjC)-domain-containing proteins and discuss their cellular functions in relation to their potential enzymatic activities.     

Chromatin remodelling in mammalian cells by ISWI-type complexes--where, when and why?

The specific location of nucleosomes on DNA has important inhibitory or activating roles in the regulation of DNA-dependent processes as it affects the DNA accessibility. Nucleosome positions depend on the ATP-coupled activity of chromatin-remodelling complexes that translocate nucleosomes or evict them from the DNA. The mammalian cell harbors numerous different remodelling complexes that possess distinct activities. These can translate a variety of signals into certain patterns of nucleosome positions with specific functions. Although chromatin remodellers have been extensively studied in vitro, much less is known about how they operate in their cellular environment. Here, we review the cellular activities of the mammalian imitation switch proteins and discuss mechanisms by which they are targeted to sites where their activity is needed.     

Transcellular proteolysis demonstrated by novel cell surface-associated substrates of dipeptidyl peptidase IV (CD26)

Proteolytic enzymes contribute to the regulation of cellular functions such as cell proliferation and death, cytokine production, and matrix remodeling. Dipeptidyl peptidase IV (DP IV) catalyzes the cleavage of several cytokines and thereby contributes to the regulation of cytokine production and the proliferation of immune cells. Here we show for the first time that cell surface-bound DP IV catalyzes the cleavage of specific substrates that are associated with the cellular surface of neighboring cells. Rhodamine 110 (R110), a highly fluorescent xanthene dye, was used to synthesize dipeptidyl peptidase IV (DP IV/CD26) substrates Gly(Ala)-Pro-R110-R, thus facilitating a stable binding of the fluorescent moiety on the cell surface. The fixation resulted from the interaction with the reactive anchor rhodamine and allowed the quantification of cellular DP IV activity on single cells. The reactivity, length, and hydrophobicity of rhodamine was characterized as the decisive factor that facilitated the determination of cellular DP IV activity. Using fluorescence microscopy, it was possible to differentiate between different DP IV activities. The hydrolysis of cell-bound substrates Xaa-Pro-R110-R by DP IV of neighboring cells and by soluble DP IV was shown using flow cytometry. These data demonstrate that ectopeptidases such as DP IV may be involved in communication between blood cells via proteolysis of cell-associated substrates.     

Characterization of laminin 5B and NH2-terminal proteolytic fragment of its alpha3B chain: promotion of cellular adhesion, migration, and proliferation

Various laminin isoforms have specific biological functions depending on their structures. Laminin 5A, which consists of the three truncated chains alpha3A, beta3, and gamma2, is known to have strong activity to promote cell adhesion and migration, whereas a laminin 5 variant consisting of a full-sized alpha3 chain (alpha3Beta) and the beta3 and gamma2 chains, laminin 5B, has not been characterized yet. In the present study, we for the first time cloned a full-length human laminin alpha3B cDNA and isolated the human laminin 5B protein. The molecular size of the mature alpha3B chain (335 kDa) was approximately twice as large as the mature alpha3A chain in laminin 5A. Laminin 5B had significantly higher cell adhesion and cell migration activities than laminin 5A. In addition, laminin 5B potently stimulated cell proliferation when added into the culture medium directly. Furthermore, we found that the alpha3B chain undergoes proteolytic cleavage releasing a 190-kDa NH(2)-terminal fragment. The 190-kDa fragment had activities to promote cellular adhesion, migration, and proliferation through its interaction with integrin alpha(3)beta(1). These activities of the NH(2)-terminal structure of the alpha3B chain seem to contribute to the prominent biological activities and the physiological functions of laminin 5B.