Epidermal Growth Factor Receptor Phosphorylation Sites Ser991 and Tyr998 Are Implicated in the Regulation of Receptor Endocytosis and Phosphorylations at Ser1039 and Thr1041

Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser⁹⁹¹ and Tyr⁹⁹⁸ accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195–4206). EGF-induced phosphorylations at Ser¹⁰³⁹ and Thr¹⁰⁴¹ were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr⁹⁹⁸, Ser⁹⁹¹, Ser¹⁰³⁹, and Thr¹⁰⁴¹ governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.Upon activation by ligand, the epidermal growth factor receptor (EGFR)¹ dimerizes, sometimes as heterodimers with other EGFR family members; is catalytically activated by reorientation of kinase region subdomains; becomes covalently modified by phosphorylation and ubiquitination; and interacts with a variety of intracellular proteins (1, 2). These events activate intracellular signaling cascades, and concurrently the dimerized receptors become internalized through endocytosis and then may be recycled to the cell surface or degraded in lysosomes (3). Systematic analysis of EGFR family phosphorylation-dependent protein interactions has been assessed (4, 5), and many of the known EGFR-interacting proteins can be categorized as functioning in cellular processes such as EGF-induced signal transduction and EGFR endocytosis and trafficking. Temporal analysis of tyrosine phosphorylation following EGF treatment of cells revealed groups of EGFR substrates with shared profiles of phosphorylation kinetics, including some that display rapid kinetics of phosphorylation accumulation and are involved in signal transduction (e.g. ERK kinase activation) and others that accumulate more slowly following ligand treatment and are involved in receptor internalization and down-regulation (5–11). Although advances in MS and the definition of phosphorylation-dependent protein-protein interactions have led to a greatly expanded view of EGFR function and regulation, our understanding of the biological consequences and spatial-temporal relationships of individual modifications is incomplete.In a previous quantitative phosphoproteomics study aimed at the identification of drug-modulated changes in phosphorylation associated with the EGFR network, a cluster of three sites of phosphorylation in the EGFR carboxyl tail region was identified as affected by receptor stimulation by EGF and inhibited by the ATP-competitive EGFR inhibitor PKI166 in human A431 tumor cells and xenograft tumors (12). The three sites in the cluster, Ser⁹⁹¹,² Ser⁹⁹⁵, and Tyr⁹⁹⁸, are localized within a single tryptic peptide having the sequence MHLPSPTDSNFYR that spans residues 987–999. The phosphorylation of Tyr⁹⁹⁸ was first described by Stover et al. (12), whereas the two serine sites were shown previously to be phosphorylated by Heisermann and Gill (13). Numerous recent studies using different cultured cell models have verified the phosphorylation of EGFR at Tyr⁹⁹⁸ and Ser⁹⁹¹ (10, 11, 14, 15), and Thr⁹⁹³ was also observed to be phosphorylated within this same region of the EGFR in EGF-stimulated HeLa cells (10). The modulation of these sites by EGF and the EGFR inhibitor implicates them in EGFR signaling and suggests that they may have utility as pharmacodynamic markers of EGFR activity. However, the function and importance of these sites, their modulation by kinases and phosphatases, and possible roles in EGFR function remain unknown.Several amino acid residues in the EGFR have been implicated in the regulation of its trafficking. Sorkin et al. (16) showed that substitution of Tyr⁹⁹⁸ with phenylalanine rendered high density EGFRs defective for endocytosis and interaction with AP-2. More recent kinetic studies using MS indicated that EGFR phosphorylation at both Tyr⁹⁹⁸ (5) and Ser⁹⁹¹ (10) occurs relatively slowly compared with other EGF-induced tyrosine phosphorylations known to be involved in receptor-proximal signal transduction. For example, Mann and co-workers (10) recorded maximal phosphorylation at EGFR sites Tyr¹¹¹⁰, Tyr¹¹⁷², and Tyr¹¹⁹⁷ at 1 min post-EGF, whereas EGF-stimulated phosphorylation at Tyr⁹⁹⁸ was still increasing at 15 min post-EGF (5), and a peptide containing both Ser(P)⁹⁹¹ and Thr(P)⁹⁹³ peaked after 10 min (10). However, the role of phosphorylation at Tyr⁹⁹⁸ and Ser⁹⁹¹ has not been reported. Another region of the EGFR, spanning residues 1026–1046, was identified by Zwang and Yarden (17) as a target of phosphorylation downstream of the stress-activated mitogen-activated protein (MAP) kinase p38 and associated with transient internalization and recycling of the EGFR in response to cytokine (TNFα) and stress challenges such as UV irradiation and the chemotherapeutic agent cisplatinum. Within this part of the receptor, a 13-residue section spanning 1029–1041 and the leucines at 1034 and 1035 in particular were found to be essential for ligand- and dimerization-induced EGFR endocytosis (18). Although both EGF- and stress-induced EGFR internalization may be clathrin-mediated, they differ in that the former leads to receptor down-regulation and involves the E3 ubiquitin ligase CBL (19), whereas the latter involves receptor recycling, is not associated with receptor phosphorylation at the CBL binding site Tyr(P)¹⁰⁶⁹, and, in the case of TNFα treatment, involves activation of the transforming growth factor β-activated kinase TAK1 upstream of p38 (20). Interestingly although p38 kinase is not required for EGF-induced EGFR internalization, it is required for CBL-dependent receptor degradation (21). Therefore, alternate pathways involving p38 kinase regulate the down-regulation or recycling of the EGFR in response to diverse extracellular signals. However, the molecular details that govern these two processes are not fully understood.In the current study, EGFR phosphorylation, signaling, protein-protein interactions, and trafficking were analyzed to address the role of Tyr⁹⁹⁸ and Ser⁹⁹¹ in EGFR endocytosis. This was achieved by application of complementary methods including quantitative selected reaction monitoring (SRM, also known as MRM for multiple reaction monitoring) mass spectrometry, fluorescence imaging and cell sorting, immunoaffinity protein enrichment and blotting, and site-directed mutagenesis. Substitution mutations that prevented phosphorylation at EGFR Tyr⁹⁹⁸ and Ser⁹⁹¹ did not prevent EGFR-to-ERK signaling but impaired EGF-induced receptor internalization and stimulated p38 kinase-dependent receptor phosphorylation at positions Ser¹⁰³⁹ and Thr¹⁰⁴¹. These findings confirm the importance of Tyr⁹⁹⁸ and reveal a role for Ser⁹⁹¹ in EGF-mediated EGFR internalization possibly involving cross-talk with the p38 kinase-dependent EGFR recycling pathway.     

Convergent evolution, habitat shifts and variable diversification rates in the ovenbird-woodcreeper family (Furnariidae)

Background  

The Neotropical ovenbird-woodcreeper family (Furnariidae) is an avian group characterized by exceptionally diverse ecomorphological adaptations. For instance, members of the family are known to construct nests of a remarkable variety. This offers a unique opportunity to examine whether changes in nest design, accompanied by expansions into new habitats, facilitates diversification. We present a multi-gene phylogeny and age estimates for the ovenbird-woodcreeper family and use these results to estimate the degree of convergent evolution in both phenotype and habitat utilisation. Furthermore, we discuss whether variation in species richness among ovenbird clades could be explained by differences in clade-specific diversification rates, and whether these rates differ among lineages with different nesting habits. In addition, the systematic positions of some enigmatic ovenbird taxa and the postulated monophyly of some species-rich genera are evaluated.     

ArfGAP1 interacts with coat proteins through tryptophan-based motifs

The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a ³²⁹WETF sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide (⁴⁰⁵AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the δ-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.     

High allelic variation of MHC class II alpha antigen and the role of selection in wild and cultured Sparus aurata populations

Genetic polymorphism and differentiation in wild and cultured sea bream samples were studied after amplification, cloning, and partial sequence of the major histocompatibility complex (MHC) class II alpha antigen. Forty-one alleles were detected from 43 unrelated individuals and sequence alignment of the obtained alleles revealed 28 polymorphic sites. High heterozygosity values and allelic richness were unveiled for both wild and cultured populations. The substitution pattern (d_N /d_S = 0.7) is not consistent with the effect of diversifying selection, indicating lower selection pressure on the a2 domain, as well as that too few advantageous non-synonymous mutations have accumulated as substrate for the diversifying selection to act. Comparison with previously published results on microsatellite markers suggests that balancing selection in MHC genes reduces the genetic drift and bottleneck effects that are common in aquaculture and which are known to reduce genetic variation at neutral markers. The present study stresses that both coding and non-coding loci should be analyzed for designing proper management strategies.     

Chromosome Stability and Gene Loss in Cockroach Endosymbionts

Insect endosymbiont genomes reflect massive gene loss. Two Blattabacterium genomes display colinearity and similar gene contents, despite high orthologous gene divergence, reflecting over 140 million years of independent evolution in separate cockroach lineages. We speculate that distant homologs may replace the functions of some eliminated genes through broadened substrate specificity.Obligate symbionts of insects exhibit extreme patterns of genome evolution and include the smallest known bacterial genomes (10, 11, 14). Two recently published sequences of Blattabacterium, the obligate symbiont of cockroaches (7, 16), present the opportunity to analyze genome evolution in an additional symbiont lineage with extreme genome reduction.     

Temperature-dependent phenotypic variation of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> lipooligosaccharides

Background  

Campylobacter jejuni is a major bacterial cause of food-borne enteritis, and its lipooligosaccharide (LOS) plays an initiating role in the development of the autoimmune neuropathy, Guillain-Barré syndrome, by induction of anti-neural cross-reactive antibodies through ganglioside molecular mimicry.