SYK

"Cystic Fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the gene that encodes for the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). With more than 1600 mutations
reported, a single mutation– the deletion of phenylalanine residue at position 508 – accounts for more than 70% of chromosomes worldwide. The presence of this mutation causes CFTR retention at the endoplasmic reticulum (ER) and its early degradation at the proteasome.
We have studied the effect of AMPK phosphorylation on a CFTR
variant (S573C) found in non-CF patients with pancreatitis. This variant was found to be less functional than wt-CFTR and more sensitive to AMPK phosphorylation. Our results suggest that S573 plays an important role in channel gating.
We have also studied the effect of CK2 phosphorylation upon
CFTR function and found that TBB inhibits wt-CFTR currents without affecting F508del-CFTR. Further insight was achieved by mutating three putative CK2-phosphorylation sites. (S422, S511 and T1471). Mutating S511 (A or D) did not have any detectable effect upon wt-CFTR function, while mutating S422 (A or D) significantly , decreases or increases CFTR function, respectively. Both T1471A- and T1471DCFTR are less functional than wt-CFTR, and significantly inhibitable by TBB, similarly to S422 mutants. These data suggest that CK2 may be a positive effector for CFTR biogenesis, trafficking or function.
Mutating Y512, the SYK consensus phosphorylation site, into an A results in a significant increase in CFTR function in both wt- and F508del backgrounds. This mutation may cause a conformational change that favours channel gating.
Altogether, these data provide novel insight into CFTR regulation
by phosphorylation and thus to our understanding of the molecular mechanisms that govern its trafficking and/or function.


Key words: CFTR, Cystic Fibrosis, Phosphorylation, CK2, SYK,
AMPK, Metformin"

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.

Background Mantle cell lymphoma (MCL) is currently an incurable entity, and new therapeutic approaches are needed. We have applied a high-throughput phospho-proteomic technique to MCL cell lines to identify activated pathways and we have then validated our data in both cell lines and tumor tissues. Methods PhosphoScan analysis was performed on MCL cell lines. Results were validated by flow cytometry and western blotting. Functional validation was performed by blocking the most active pathway in MCL cell lines. Results PhosphoScan identified more than 300 tyrosine-phosporylated proteins, among which many protein kinases. The most abundant peptides belonged to proteins connected with B-cell receptor (BCR) signaling. Active BCR signaling was demonstrated by flow cytometry in MCL cells and by western blotting in MCL tumor tissues. Blocking BCR signaling by Syk inhibitor piceatannol induced dose/time-dependent apoptosis in MCL cell lines, as well as several modifications in the phosphorylation status of BCR pathway members and a collapse of cyclin D1 protein levels. Conclusion Our data support a pro-survival role of BCR signaling in MCL and suggest that this pathway might be a candidate for therapy. Our findings also suggest that Syk activation patterns might be different in MCL compared to other lymphoma subtypes.

Among peripheral T-cell lymphomas (PTCL), the heterogeneous category of unspecified PTCL represents the most common subtype. Nevertheless, recurrent chromosomal translocations are unknown in this aggressive type of lymphoma. Here we describe a novel t(5;9)(q33;q22) in unspecified PTCL. Molecular analyses delineated the breakpoints to ITK and SYK resulting in a previously undescribed expression of the Syk tyrosine kinase by Itk. ITK–SYK

Airway remodeling is not specifically targeted by current asthma medications, partly owing to the lack of understanding of remodeling mechanisms, altogether posing great challenges in asthma treatment. Increased airway smooth muscle (ASM) mass due to hyperplasia/hypertrophy contributes significantly to overall airway remodeling and correlates with decline in lung function. Recent evidence suggests that IgE sensitization can enhance the survival and mediator release in inflammatory cells. Human ASM (HASM) cells express both low affinity (FcεRII/CD23) and high affinity IgE Fc receptors (FcεRI), and IgE can modulate the contractile and synthetic function of HASM cells. IgE was recently shown to induce HASM cell proliferation but the detailed mechanisms remain unknown. We report here that IgE sensitization induces HASM cell proliferation, as measured by 3H-thymidine, EdU incorporation, and manual cell counting. As an upstream signature component of FcεRI signaling, inhibition of spleen ...

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regulation is associated with a variety of allergic disorders and autoimmune diseases. To identify small molecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulk quantity. We studied the expression of recombinant human Syk in three heterologous systems: E. coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium discoideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, whereas in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain protein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor. Molecular modeling and docking studies were performed to understand the binding mode and critical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinase domain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levels of tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we conclude that BEVS is the ideal host to produce an active and stable enzyme, which can be successfully employed for screening of Syk inhibitors in a high-throughput system.