Negative feedback regulation of the tumor suppressor PTEN by phosphoinositide-induced serine phosphorylation

The PTEN tumor suppressor phosphatase directly counteracts the multiple functions of phosphatidylinositol 3-kinase by removing phosphate from the D3 position of inositol phospholipids. Like many lymphomas and leukemias, the Jurkat T cell line lacks PTEN protein due to frame-shift mutations in both PTEN alleles and therefore survives in long-term cell culture. We report that PTEN reintroduced into Jurkat was highly phosphorylated on serines 380 and 385 in its C terminus, particularly the former site. Phosphate was also detected at Ser(380) in PTEN in untransformed human T cells. Treatments that reduced the levels of D3-phospholipids in the cells resulted in reduced phosphorylation and accelerated degradation of PTEN. In contrast, expression of inactive PTEN-C124G or coexpression of a constitutively active protein kinase B led to increased phosphorylation and slower degradation of PTEN. These results suggest that PTEN normally is subjected to a feedback mechanism of regulation aimed at maintaining homeostatic levels of D3-phosphoinositides, which are crucial for T cell survival and activation.     

Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish

L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with reduced Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.     

BRCA1 phosphorylation by Aurora-A in the regulation of G2 to M transition

Aurora-A/BTAK/STK15 localizes to the centrosome in the G(2)-M phase, and its kinase activity regulates the G(2) to M transition of the cell cycle. Previous studies have shown that the BRCA1 breast cancer tumor suppressor also localizes to the centrosome and that BRCA1 inactivation results in loss of the G(2)-M checkpoint. We demonstrate here that Aurora-A physically binds to and phosphorylates BRCA1. Biochemical analysis showed that BRCA1 amino acids 1314-1863 binds to Aurora-A. Site-directed mutagenesis indicated that Ser(308) of BRCA1 is phosphorylated by Aurora-A in vitro. Anti-phospho-specific antibodies against Ser(308) of BRCA1 demonstrated that Ser(308) is phosphorylated in vivo. Phosphorylation of Ser(308) increased in the early M phase when Aurora-A activity also increases; these effects could be abolished by ionizing radiation. Consistent with these observations, acute loss of Aurora-A by small interfering RNA resulted in reduced phosphorylation of BRCA1 Ser(308), and transient infection of adenovirus Aurora-A increased Ser(308) phosphorylation. Mutation of a single phosphorylation site of BRCA1 (S308N), when expressed in BRCA1-deficient mouse embryo fibroblasts, decreased the number of cells in the M phase to a degree similar to that with wild type BRCA1-mediated G(2) arrest induced by DNA damage. We propose that BRCA1 phosphorylation by Aurora-A plays a role in G(2) to M transition of cell cycle.     

Negative regulation of the E3 ubiquitin ligase itch via Fyn-mediated tyrosine phosphorylation

Conjugation of ubiquitin (Ub) to a protein substrate targets the substrate for degradation or functional modification, which is tightly controlled by diverse mechanisms including phosphorylation of the substrate. An emerging mechanism involves regulation of the E3 Ub ligase, for example, the JNK-dependent phosphorylation and activation of Itch E3 ligase, which controls the turnover of Jun proteins and T cell differentiation. Here we show that Itch is also modulated by an Src kinase Fyn via tyrosine phosphorylation at the Tyr371 residue. Fyn associates with Itch, and loss of Fyn results in reduced Itch phosphorylation. Importantly, tyrosine phosphorylation of Itch appears to reduce its interaction with its substrate JunB. The turnover of JunB is accelerated in Fyn-deficient T cells, which is further reconstituted by Itch Tyr371 mutation. Thus, in contrast to the activation pathway mediated by serine/threonine phosphorylation, tyrosine phosphorylation of Itch plays a negative role in modulating Itch-promoted ubiquitination.     

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca(2+) mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca(2+)](cyt), production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca(2+) transients mainly due to plasma membrane Ca(2+) influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca(2+)](cyt) elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca(2+) mobilization and may play a crucial role in temporal regulation of [Ca(2+)](cyt) signature.     

Negative regulation of Raf-1 by phosphorylation of serine 621.

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.     

Negative regulation of condensin I by CK2-mediated phosphorylation

Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.     

Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT

Mitogen-activated protein kinase (MAPK) cascades are involved in a variety of cellular responses including proliferation, differentiation, and apoptosis. We have developed an expression screening method to detect in vivo substrates of MAPKs in mammalian cells, and identified a membrane protein, linker for activation of T cells (LAT), as an MAPK target. LAT, an adapter protein essential for T-cell signaling, is phosphorylated at its Thr 155 by ERK in response to T-cell receptor stimulation. Thr 155 phosphorylation reduces the ability of LAT to recruit PLCgamma1 and SLP76, leading to attenuation of subsequent downstream events such as [Ca2+]i mobilization and activation of the ERK pathway. Our data reveal a new role for MAPKs in a negative feedback loop in T-cell activation via threonine phosphorylation of LAT.     

Negative feedback regulation among SR splicing factors encoded by Rbp1 and Rbp1-like in Drosophila

SR proteins constitute a widely conserved family of splicing regulators. Negative autoregulation of SR proteins has been proposed to exert homeostatic control on the splicing environment, but few examples have been studied and the role of isoforms that lack the RS domain is unclear. We show that genes Rbp1 and Rbp1-like, which encode Drosophila homologs of mammalian SRp20, negatively autoregulate and crossregulate at the level of alternative 3' splice site selection. This adjusts the relative expression of isoforms with either an RS domain or unrelated C-terminal domains (ALT) that are rich in serine and threonine. The effects of RBP1-ALT on splicing of doublesex and Rbp1-like are opposite to those of RBP1-RS and RBP1L-RS. RBP1-ALT and -RS exert opposing negative feedback on the ALT/RS ratio. However, RBP1-ALT inhibits the expression of RBP1-RS while stimulating that of RBP1L-RS. This asymmetry may contribute to changes in the RBP1-RS/RBP1L-RS ratio that are observed during development. These results provide the first example of a feedback-regulated SR protein network with evidence of an active homeostatic role for alternative isoforms.