Tyrosine phosphorylation directs TACE into extracellular vesicles via unconventional secretion

When studying how HIV‐1 Nef can promote packaging of the proinflammatory transmembrane protease TACE (tumor necrosis factor‐α converting enzyme) into extracellular vesicles (EVs) we have revealed a novel tyrosine kinase‐regulated unconventional protein secretion (UPS) pathway for TACE. When TACE was expressed without its trafficking cofactor iRhom allosteric Hck activation by Nef triggered translocation of TACE into EVs. This process was insensitive to blocking of classical secretion by inhibiting endoplasmic reticulum (ER) to Golgi transport, and involved a distinct form of TACE devoid of normal glycosylation and incompletely processed for prodomain removal. Like most other examples of UPS this process was Golgi reassembly stacking protein (GRASP)‐dependent but was not associated with ER stress. These data indicate that Hck‐activated UPS provides an alternative pathway for TACE secretion that can bypass iRhom‐dependent ER to Golgi transfer, and suggest that tyrosine phosphorylation might have a more general role in regulating UPS.      

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10(-7)M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein-tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.     

Association of Hck genetic polymorphisms with gene expression and COPD

Polymorphonuclear leukocytes (PMNs) are major effector cells in the chronic airway inflammation in chronic obstructive pulmonary disease (COPD). PMN degranulation is associated with degradation of extracellular matrix and tissue damage. Hck is an essential molecule in the signaling pathway regulating PMN degranulation. We hypothesized that polymorphisms affect the expression level of Hck, which, in turn, modulates PMN mediator release and tissue damage and influences the development of COPD. Here we systematically investigated genetic tag polymorphisms of the Hck gene, Hck mRNA and protein expression pattern in PMNs, and PMN mediator release (myeloperoxidase) in 60 healthy white subjects, and assessed their association with the use of several genetic models. The association of genetic polymorphisms with COPD-related phenotypes was determined in the lung healthy study cohort (LHS). We identified a novel 15 bp insertion/deletion polymorphism (8,656 L/S) in intron 1 of the Hck gene, which was associated with differential expression of Hck protein and PMN myeloperoxidase release. In the LHS cohort, there was significant interaction between the 8,656 L/S polymorphism and smoking on baseline lung function and 8,656 L/S was associated with bronchodilator response. These data suggest that the insertion/deletion polymorphism could be a functional polymorphism of the Hck gene, may contribute to COPD pathogenesis and modify COPD-related phenotypes.     

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.     

Macrophage mesenchymal migration requires podosome stabilization by filamin A

Filamin A (FLNa) is a cross-linker of actin filaments and serves as a scaffold protein mostly involved in the regulation of actin polymerization. It is distributed ubiquitously, and null mutations have strong consequences on embryonic development in humans, with organ defects which suggest deficiencies in cell migration. We have reported previously that macrophages, the archetypal migratory cells, use the protease- and podosome-dependent mesenchymal migration mode in dense three-dimensional environments, whereas they use the protease- and podosome-independent amoeboid mode in more porous matrices. Because FLNa has been shown to localize to podosomes, we hypothesized that the defects seen in patients carrying FLNa mutations could be related to the capacity of certain cell types to form podosomes. Using strategies based on FLNa knock-out, knockdown, and rescue, we show that FLNa (i) is involved in podosome stability and their organization as rosettes and three-dimensional podosomes, (ii) regulates the proteolysis of the matrix mediated by podosomes in macrophages, (iii) is required for podosome rosette formation triggered by Hck, and (iv) is necessary for mesenchymal migration but dispensable for amoeboid migration. These new functions assigned to FLNa, particularly its role in mesenchymal migration, could be directly related to the defects in cell migration described during the embryonic development in FLNa-defective patients.     

Improved 3D gd-HCACO and gd-(H)CACO-TOCSY experiments for isotopically enriched proteins dissolved in H2O

Summary Pulsed field gradients were incorporated into the HCACO experiment for acquiring spectra on isotopically enriched protein samples dissolved in H₂O. Excellent water suppression and spectral quality were achieved using the modified pulse sequence (gd-HCACO), as demonstrated for a ¹³C-/¹⁵N-labeled sample of the SH2 domain from the hematopoietic cellular kinase dissolved in 90% H₂O/10% D₂O. Strong correlations for all residues were observed in the gd-HCACO spectrum, even for residues having -protons resonating exactly at the H₂O frequency. The HCACO-TOCSY experiment was modified to correlate intraresidue ¹³C (rather than ¹H), carbonyl (¹³C), and aliphatic side-chain protons [(H)CACO-TOCSY]. Pulsed field gradients were also incorporated into the (H)CACO-TOCSY experiment for water suppression.     

An examination of dynamics crosstalk between SH2 and SH3 domains by hydrogen/deuterium exchange and mass spectrometry

The ability of proteins to regulate their own enzymatic activity can be facilitated by changes in structure or protein dynamics in response to external regulators. Because many proteins contain SH2 and SH3 domains, transmission of information between the domains is a potential method of allosteric regulation. To determine if ligand binding to one modular domain may alter structural dynamics in an adjacent domain, allowing potential transmission of information through the protein, we used hydrogen exchange and mass spectrometry to measure changes in protein dynamics in the SH3 and SH2 domains of hematopoietic cell kinase (Hck). Ligand binding to either domain had little or no effect on hydrogen exchange in the adjacent domain, suggesting that changes in protein structure or dynamics are not a means of SH2/SH3 crosstalk. Furthermore, ligands of varying affinity covalently attached to SH3/SH2 altered dynamics only in the domain to which they bind. Such results demonstrate that ligand binding may not structurally alter adjacent SH3/SH2 domains and implies that other aspects of protein architecture contribute to the multiple levels of regulation in proteins containing SH3 and SH2 domains.     

Versatile retargeting of SH3 domain binding by modification of non-conserved loop residues

Src-homology (SH3) domain belongs to a class of ubiquitous modular protein domains found in nature. SH3 domains have a conserved surface that recognises proline-rich peptides in ligand proteins, but additional contacts also contribute to binding. Using the SH3 domain of hematopoietic cell kinase as a test case, we show that SH3 binding properties can be profoundly altered by modifications within a hexapeptide sequence in the RT-loop region that is not involved in recognition of currently known consensus SH3 target peptides. These results highlight the role of non-conserved regions in SH3 target selection, and introduce a strategy that may be generally feasible for generating artificial SH3 domains with desired ligand binding properties.