Laminin-1 is phosphorylated by ecto-protein kinases of monocytes

Monocytes encounter basement membranes and interact with laminins while crossing the vascular barrier. It is known that these cells possess ecto-protein kinase activity on their surface. Several proteins of the extracellular matrix can be phosphorylated by ectokinases. Therefore, it has been hypothesized that monocyte ectokinases could phosphorylate laminins and influence their biological properties. In order to test the above hypothesis, we used intact human monocytes and adenosine triphosphate labeled with radioactive phosphate at the third phosphate ([gamma-32P]-ATP) to phosphorylate laminin-1. Autoradiography after sodium dodecyl sulphate polyacrylamyde gel electrophoresis (SDS-PAGE) electrophoresis indicated phosphorylation of laminin-1 on the beta and/or gamma chains. After phosphorylation, phosphoserine could be detected on Western blots by a specific monoclonal antibody. Phosphorylation was not detected when monocytes were pre-treated with trypsin and was inhibited by a specific ecto-protein kinase inhibitor (K252b). Laminin phosphorylation was also inhibited by heparin, a known inhibitor of casein kinase II and by pretreatment of monocytes by a monoclonal anti-casein kinase II antibody. Heparin binding, cell attachment and proliferation, and monocyte migration were enhanced on the phosphorylated laminin-1 as compared to the non-phosphorylated controls. These data indicate that laminin-1 can be phosphorylated by monocyte casein kinase II type ectokinase. This phosphorylation influences important functions of laminin and therefore could provide an additional means for the interaction of monocytes with basement membranes.     

Atrial natriuretic peptide is phosphorylated by intact cells through cAMP-dependent ecto-protein kinase.

Recently we demonstrated the presence of cell-surface-located cAMP-dependent protein kinase (ecto-PK A) activity in a number of different cell types [Kübler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J. and Kinzel, V. (1989) J. Biol. Chem. 264, 14549-14555]. The question of the physiological role of externally directed kinase activity prompted a search for potential natural substrates present in the intercellular fluid. In the present study we have investigated the phosphorylation by ecto-PK A of the human atrial natriuretic peptide ANP99-126, a hormone released by cardiac cells. This 28-amino-acid peptide carries the phosphorylation consensus sequence Arg-Arg-Ser-Ser for the PK A. Incubation of various cell lines (including epithelial, epidermal, myoblast and lymphoma cells) or freshly isolated blood cells (macrophages, erythrocytes and platelets) with ANP in the presence of low micromolar concentrations of ATP resulted in the phosphorylation of ANP at Ser residues. The ANP phosphorylation reaction proved strictly dependent on cAMP; cAMP could not be replaced by cGMP. The phosphorylation was inhibited by the PK A-specific inhibitory peptide and increased linearily for up to 15 min and with a Km value of 3-5 microM for ANP. At higher ATP concentrations (greater than 100 microM) the incorporation rates amounted to about 0.3 mmol P (mol ANP)-1 min-1. The rise of intracellular cAMP in HEL30 (an epidermal cell line) after application of the beta-adrenergic receptor agonist isoproterenol led to an approximately three-fold stimulation of ANP phosphorylation which appears to be brought about by an efflux of intracellular cAMP. Employing cell supernatant fluids and cell sonicates, it could be shown that the phosphorylation of ANP results from the ecto-PK A. Comparison of ANP with ANP phosphorylated in vitro using purified catalytic subunit of PK A showed that phosphorylation is accompanied by certain changes in the average solution conformation of the peptide, consistent with the changes known to occur in its biological activity. Our results demonstrate cAMP-dependent phosphorylation of the peptide hormone analogue ANP99-126 by intact cells through ecto-PK A, an intriguing mechanism for post-translational processing of ANP.     

Cbl-associated protein is tyrosine phosphorylated by c-Abl and c-Src kinases

Background  

The c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases

alpha-Synuclein (alpha-Syn) is implicated in the pathogenesis of Parkinson's Disease, genetically through missense mutations linked to early onset disease and pathologically through its presence in Lewy bodies. alpha-Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha-Syn has not been established (, ). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of alpha-Syn are conserved in all orthologs and beta-Syn paralogs described to date, suggesting that these residues may be of functional importance (). For this reason, experiments were performed to determine whether alpha-Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, alpha-Syn is phosphorylated within 2 min of pervanadate treatment in alpha-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function (). Finally, we demonstrate that alpha-Syn can be phosphorylated directly both in cotransfection experiments using c-Src and Fyn expression vectors and in in vitro kinase assays with purified kinases. These data suggest that alpha-Syn can be a target for phosphorylation by the Src family of PTKs.     

Interferon production by primary culture of human endothelial cells

Endothelial cells of human umbilical vein are capable of producing interferon upon induction with Newcastle disease virus, influenza virus, and poly I: poly C, but not staphylococcal enterotoxin A. All the interferons produced belonged to the alpha-type. After treatment with influenza virus the endothelial cells produce two subtypes of alpha-interferon: acid-labile and acid-stable.     

Vitronectin in the substratum of endothelial cells is cross-linked and phosphorylated.

Vitronectin (VN), previously shown to be a substrate for purified transglutaminases, was demonstrated in this study to be cross-linked when incubated with HUVEC and EAhy926 cells. The cross-linking was calcium-dependent and required that VN be plated at the substratum of the cells. These cells also phosphorylated VN, but in contrast to a previous study demonstrating a cAMP-dependent protein kinase in platelets, the phosphorylation of VN by was decreased with the addition of 1mM cAMP. The cross-linking of VN by endothelial cells demonstrates that the adhesion of these cells to VN is a dynamic process in which the substratum may be enzymatically altered. Furthermore, the modifications of VN by cross-linking and phosphorylation could modulate the functions of VN and influence events such as endothelial cell proliferation and angiogenesis.     

Interferon gamma priming is not critical for IL-12 production of murine spleen cells

Interferon gamma (IFN-gamma) priming is considered to be critical for interleukin 12 (IL-12) production of murine macrophages and human monocytes by lipopolysaccharide (LPS) stimulation. In our present experiments, freshly prepared spleen cells (f-spleen cells) were confirmed not to produce detectable level of IL-12 by LPS stimulation, although they produced significant amount of IL-12 by the stimulation with LPS plus IFN-gamma. However, the stimulation only with LPS induced IL-12 production of spleen cells preincubated in the absence of IFN-gamma. Findings on IL-12 p40 mRNA accumulation were consistent with their IL-12 production. Essentially the same results were obtained using spleen cells from IFN-gamma deficient mice. In the presence of anti-IL-10, f-spleen cells produced IL-12 upon LPS stimulation, indicating that the failure of f-spleen cells in IL-12 production is caused by IL-10 produced by themselves upon LPS stimulation. In addition, f-spleen cells produced IL-12 upon CD40 ligand stimulation, and the production was hardly affected by the presence of IFN-gamma or preincubation. These results indicate that IFN-gamma priming is not critical for IL-12 production of spleen cells stimulated with LPS or CD40 ligand, although IFN-gamma enhances the production, especially, in response to LPS stimulation.     

Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro.

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.     

Transferrin is made and bound by photoreceptor cells

Retinal pigment epithelial cells, which form one aspect of the blood-retinal barrier, control the access of blood-borne components such as diferric transferrin to the neural retina. It has recently been shown that RPE cells remove iron from diferric transferrin in a low pH compartment and subsequently release it in a low molecular weight form that can be chelated by apo-transferrin (Hunt and Davis: J. Cell Physiol. 152:102–110, 1992). It is now shown that photoreceptor cells can bind diferric transferrin to receptors on their inner segments. Moreover, polymerase chain reaction and in situ hybridization show that cells of the neural retina, particularly photoreceptors, make apo-transferrin. © 1993 Wiley-Liss, Inc.     

The mobile ferrous iron pool in Escherichia coli is bound to a phosphorylated sugar derivative

Based on in vivo Mössbauer spectroscopy it has previously been demonstrated that the intracellular iron pool of Escherichia coli, grown in iron deficient media supplemented with siderophores as the sole iron source, is dominated by a single Fe²⁺ and a single Fe³⁺ species. We have isolated the ferrous ion species and have purified it employing native column PAGE, chromatography and ultrafiltration. The purified compound displays an M _app of 2.2 kDa and an extremely low isoelectric point (pI) of 1.05. It is shown that this ferrous ion binding compound is neither a protein nor a nucleotide, rather it is composed mainly of phosphorylated sugar derivatives. This compound binds approximately 40% of the cytoplasmic iron. Therefore it is proposed that this oligomeric ferrous carbohydrate phosphate represents the long sought after mobile, low molecular mass iron pool.     

The prion-like domain of Fused in Sarcoma is phosphorylated by multiple kinases affecting liquid- and solid-phase transitions

Fused in Sarcoma (FUS) is a ubiquitously expressed protein that can phase-separate from nucleoplasm and cytoplasm into distinct liquid-droplet structures. It is predominantly nuclear and most of its functions are related to RNA and DNA metabolism. Excessive persistence of FUS within cytoplasmic phase-separated assemblies is implicated in the diseases amyotrophic lateral sclerosis and frontotemporal dementia. Phosphorylation of FUS’s prion-like domain (PrLD) by nuclear phosphatidylinositol 3-kinase-related kinase (PIKK)-family kinases following DNA damage was previously shown to alter FUS’s liquid-phase and solid-phase transitions in cell models and in vitro. However, proteomic data suggest that FUS’s PrLD is phosphorylated at numerous additional sites, and it is unknown if other non-PIKK and nonnuclear kinases might be influencing FUS’s phase transitions. Here we evaluate disease mutations and stress conditions that increase FUS accumulation into cytoplasmic phase-separated structures. We observed that cytoplasmic liquid-phase structures contain FUS phosphorylated at novel sites, which occurred independent of PIKK-family kinases. We engineered phosphomimetic substitutions within FUS’s PrLD and observed that mimicking a few phosphorylation sites strongly inhibited FUS solid-phase aggregation, while minimally altering liquid-phase condensation. These effects occurred independent of the exact location of the phosphomimetic substitutions, suggesting that modulation of PrLD phosphorylation may offer therapeutic strategies that are specific for solid-phase aggregation observed in disease.     

Par6 is phosphorylated by aPKC to facilitate EMT

The conserved polarity proteins Par6 and aPKC regulate cell polarization processes. However, increasing evidence also suggests that they play a role in oncogenic progression. During tumor progression, epithelial to mesenchymal transition (EMT) delineates an evolutionary conserved process that converts stationary epithelial cells into mesenchymal cells, which have an acquired ability for independent migration and invasion. In addition to signaling pathways that alter genetic programes that trigger the loss of cell-cell adhesion, alternative pathways can alter cell plasticity to regulate cell-cell cohesion and increase invasive potential. One such pathway involves TGFβ-induced phosphorylation of Par6. In epithelial cells, Par6 phosphorylation results in the dissolution of junctional complexes, cytoskeletal remodelling, and increased metastatic potential. Recently, we found that aPKC can also phosphorylate Par6 to drive EMT and increase the migratory potential of non-small cell lung cancer cells. This result has implications with respect to homeostatic and developmental processes involving polarization, and also with respect to cancer progression—particularly since aPKC has been reported to be an oncogenic regulator in various tumor cells.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.     

Early response of endothelial cells to flow is mediated by VE-cadherin

Endothelial cells are known to respond to flow onset by increasing actin turnover rate. Current models assume that an increase in the actin turnover rate should result in a rise in cell crawling speed. Here we report that confluent endothelial monolayer shows an unexpected behavior: cell crawling speed decreases by approximately 40% within the first 30 min of flow onset. A drop in crawling speed has not been observed in either subconfluent endothelial cells or in VE-cadherin-deficient cells. We found that flow onset caused an increase in the number of VE-cadherin-GFP molecules in the junctions and elicited changes in the cytoskeleton-associated fractions of alpha, beta -catenins and VE-cadherin. Flow application also increased the strength of interactions of endothelial cells with surfaces coated with recombinant VE-cadherin. These observations suggest that endothelial cell junctional proteins respond to flow transiently by increasing the strength of intercellular attachments early after flow onset and support the view on the active role of intercellular adhesions in mechanotransduction.