Christer Wernstedt

TGF-β1 binding protein: A component of the large latent complex of TGF-β1 with multiple repeat sequences

Abstract TGF-β occurs in a latent complex of high M r . We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-β1 complex, denoted TGF-β1 binding protein (TGF-β1-BP). Most of the sequence of fibroblast TGF-β1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. β-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-β1-BP purified from human platelets is considerably smaller than the fibroblast form (125–160 kd vs. 170–190 kd), suggesting that there is alternative splicing of the TGF-β1-BP gene or that TGF-β1-BP undergoes cell-specific proteolysis. TGF-β1-BP was found not to bind and inactivate TGF-β1; its role in the latent complex is discussed.

Identification of Novel Phosphorylation Sites in Hormone-sensitive Lipase That Are Phosphorylated in Response to Isoproterenol and Govern Activation Properties in Vitro

Hormone-sensitive lipase (HSL) is the rate-limiting enzyme in lipolysis. Stimulation of rat adipocytes with isoproterenol results in phosphorylation of HSL and a 50-fold increase in the rate of lipolysis. In this study, we used site-directed mutagenesis and two-dimensional phosphopeptide mapping to show that phosphorylation sites other than the previously identified Ser-563 are phosphorylated in HSL in response to isoproterenol stimulation of32P-labeled rat adipocytes. Phosphorylation of HSL in adipocytes in response to isoproterenol and in vitrophosphorylation of HSL containing Ser → Ala mutations in residues 563 and 565 (S563A,S565A) with protein kinase A (PKA), followed by tryptic phosphopeptide mapping resulted in two tryptic phosphopeptides. These tryptic phosphopeptides co-migrated with the phosphopeptides released by the same treatment of F654HPRRSSQGVLHMPLYSSPIVK675phosphorylated with PKA. Analysis of the phosphorylation site mutants, S659A, S660A, and S659A,S660A disclosed that mutagenesis of both Ser-659 and Ser-660 was necessary to abolish the activation of HSL toward a triolein substrate after phosphorylation with PKA. Mutation of Ser-563 to alanine did not cause significant change of activation compared with wild-type HSL. Hence, our results demonstrate that in addition to the previously identified Ser-563, two other PKA phosphorylation sites, Ser-659 and Ser-660, are present in HSL and, furthermore, that Ser-659 and Ser-660 are the major activity controlling sites in vitro.

Phosphorylation of Ser465 and Ser467 in the C Terminus of Smad2 Mediates Interaction with Smad4 and Is Required for Transforming Growth Factor-β Signaling

Members of the Smad family of intracellular signal transducers are essential for transforming growth factor-β (TGF-β) to exert its multifunctional effects. After activation of TGF-β receptors, Smad2 and Smad3 become phosphorylated and form heteromeric complexes with Smad4. Thereafter, these activated Smad complexes translocate to the nucleus, where they may direct transcriptional responses. Here we report that TGF-β mediates phosphorylation of Smad2 at two serine residues in the C terminus,i.e. Ser465 and Ser467, which are phosphorylated in an obligate order; phosphorylation of Ser465 requires that Ser467 be phosphorylated. Transfection of Smad2 with mutation of Ser465 and/or Ser467 to alanine residues into Mv1Lu cells resulted in dominant-negative inhibition of TGF-β signaling. These Smad2 mutants were found to stably interact with an activated TGF-β receptor complex, in contrast to wild-type Smad2, which interacts only transiently. Mutation of Ser465 and Ser467 in Smad2 abrogated complex formation of this mutant with Smad4 and blocked the nuclear accumulation not only of Smad2, but also of Smad4. Thus, heteromeric complex formation of Smad2 with Smad4 is required for nuclear translocation of Smad4. Moreover, peptides from the C terminus of Smad2 containing phosphorylated Ser465 and Ser467 were found to bind Smad4 in vitro, whereas the corresponding unphosphorylated peptides were less effective. Thus, phosphorylated Ser465 and Ser467 in Smad2 may provide a recognition site for interaction with Smad4, and phosphorylation of these sites is a key event in Smad2 activation.

Ligand-induced Vascular Endothelial Growth Factor Receptor-3 (VEGFR-3) Heterodimerization with VEGFR-2 in Primary Lymphatic Endothelial Cells Regulates Tyrosine Phosphorylation Sites

Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.

VEGF receptor-2 Y951 signaling and a role for the adapter molecule TSAd in tumor angiogenesis

Vascular endothelial growth factor receptor‐2 (VEGFR‐2) activation by VEGF‐A is essential in vasculogenesis and angiogenesis. We have generated a pan‐phosphorylation site map of VEGFR‐2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C‐terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR‐2‐expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T‐cell‐specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF‐A‐induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd‐deficient mice, indicating a critical role of Y951‐TSAd signaling in pathological angiogenesis.

Characterization of a potassium channel toxin from the Caribbean sea anemone Stichodactyla helianthus

A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.

Characterization of stable complexes involving apolipoprotein E and the amyloid β peptide in Alzheimer's disease brain

Genetic evidence suggests a role for apolipoprotein E (apoE) in Alzheimers disease (AD) amyloidogenesis. Here, amyloid-associated apoE from 32 AD patients was purified and characterized. We found that brain amyloid-associated apoE apparently exists not as free molecules but as complexes with polymers of the amyloid beta peptide (A beta). Brain A beta-apoE complexes were detected irrespective of the apoE genotype, and similar complexes could be mimicked in vitro. The fine structure of purified A beta-apoE complexes was fibrillar, and immunogold labeling revealed apoE immunoreactivity along the fibrils. Thus, we conclude that A beta-apoE complexes are principal components of AD-associated brain amyloid and that the data presented here support a role for apoE in the pathogenesis of AD.

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C‐terminal tail of the PDGF beta‐receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF‐BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C‐gamma (PLC‐gamma) compared to the wild type PDGF beta‐receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC‐gamma could be detected. These data show that tyrosine phosphorylation of PLC‐gamma is dependent on autophosphorylation of the PDGF beta‐receptor at Tyr1009 and Tyr1021.

p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils.

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor: identification of tyrosine 685 as the unique target site

Src-family tyrosine kinases are regulatory proteins that play a pivotal role in the disorganization of cadherin-dependent cell–cell contacts. We previously showed that Src was associated with vascular endothelial (VE)-cadherin and that tyrosine phosphorylation level of VE-cadherin was dramatically increased in angiogenic tissues as compared to quiescent tissues. Here, we examined whether VE-cadherin was a direct substrate for Src in vascular endothelial growth factor (VEGF)-induced VE-cadherin phosphorylation, and we identified the target tyrosine sites. Co-transfections of Chinese hamster ovary cells (CHO) cells with VE-cadherin and constitutively active Src (Y530F) resulted in a robust tyrosine phosphorylation of VE-cadherin that was not detected with kinase-dead Src (K298M). In an in vitro Src assay, the VE-cadherin cytoplasmic domain is directly phosphorylated by purified Src as well as the tyrosine residue 685 (Tyr)685-containing peptide RPSLY685AQVQ. VE-cadherin peptide mapping from human umbilical vein endothelial cells stimulated by VEGF and VE-cadherin-CHO cells transfected with active Src revealed that Y685 was the unique phosphorylated site. The presence of PhosphoY685 was confirmed by its ability to bind to C-terminal Src kinase-SH2 domain in a pull-down assay. Finally, we found that in a VEGF-induced wound-healing assay, cadherin adhesive activity was impaired by Src kinase inhibitors. These data identify that VEGF-induced-VE-cadherin tyrosine phosphorylation is mediated by Src on Y685, a process that appears to be critical for VEGF-induced endothelial cell migration.