Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils

Du, Jian; Alsayed, Yazan; Xin, Feng; Ackerman, Steven J.; Platanias, Leonidas C.

doi:10.1074/jbc.m003655200

Cited by 26 publications

(26 citation statements)

References 84 publications

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“…Western Blotting and Co-immunoprecipitations-Western blotting was performed as described previously (71,76) using either whole cell lysates or nuclear extracts from blood eosinophils, AML14.3D10 eosinophil myelocytes, and undifferentiated AML14 parental cells. HL-60 and K562 cell lines were used as positive controls as appropriate to the transcription factors being analyzed.…”

Section: Methodsmentioning

confidence: 99%

“…Antibodies used for IP and co-IP were the same as used for Western blotting. IP and co-IP were performed essentially as described previously (71,76). Briefly, the whole cell lysate from 2 to 4 ϫ 10 7 AML14.3D10 cells was precleared with 1-2 g of normal rabbit, goat, or rat affinity-purified IgG antibodies plus protein A-or G-Sepharose (Amersham Biosciences) with rotation at 4°C for 2.5 h. Antibodies were added to the precleared whole cell lysates for 2 h of rotation at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Probe sequences were as follows: dual GATA sites (5Ј-GTCCTTATCAGCCTTGCTATCTCCCT-3Ј), PU.1 site 1 (5Ј-CCCTGGGGGAAGTTCCTCCAAGGCCT-3Ј), PU.1 site 2 (5Ј-AAGTCTTTGTGAGAGGAAGCAAAGAA-3Ј), C/EBP site (5Ј-GAAGTGATGAAATGGTCC-3Ј), C/EBP and GATA-1 sites (5Ј-GAAG-TGATGAAATGGTCCTTATCAGCCT-3Ј). EMSAs were performed as described previously (71). The same antibodies used for Western blotting and co-IP were also used for supershift analyses but were supershift grade (Santa Cruz Biotechnology).…”

Section: Methodsmentioning

confidence: 99%

“…Promoter activities were determined using the Dual-luciferase Reporter Assay System (Promega) 48 h after transfection. Luciferase activities were measured as relative light units using an EG & G Berthold Lumat LB 1507 Luminometer as described previously (71). Results are reported as the mean Ϯ S.D.…”

Section: Methodsmentioning

confidence: 99%

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Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

Stankiewicz

Yang

et al. 2002

Journal of Biological Chemistry

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106

139

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GATA-1 and the ets factor PU.1 have been reported to functionally antagonize one another in the regulation of erythroid versus myeloid gene transcription and development. The CCAAT enhancer binding protein ⑀ (C/EBP⑀) is expressed as multiple isoforms and has been shown to be essential to myeloid (granulocyte) terminal differentiation. We have defined a novel synergistic, as opposed to antagonistic, combinatorial interaction between GATA-1 and PU.1, and a unique repressor role for certain C/EBP⑀ isoforms in the transcriptional regulation of a model eosinophil granulocyte gene, the major basic protein (MBP). The eosinophil-specific P2 promoter of the MBP gene contains GATA-1, C/EBP, and PU.1 consensus sites that bind these factors in nuclear extracts of the eosinophil myelocyte cell line, AML14.3D10. The promoter is transactivated by GATA-1 alone but is synergistically transactivated by low levels of PU.1 in the context of optimal levels of GATA-1. The C/EBP⑀ 27 isoform strongly represses GATA-1 activity and completely blocks GATA-1/PU.1 synergy. In vitro mutational analyses of the MBP-P2 promoter showed that both the GATA-1/PU.1 synergy, and repressor activity of C/EBP⑀ 27 are mediated via protein-protein interactions through the C/EBP and/or GATA-binding sites but not the PU.1 sites. Co-immunoprecipitations using lysates of AML14.3D10 eosinophils show that both C/EBP⑀ 32/30 and ⑀ 27 physically interact in vivo with PU.1 and GATA-1, demonstrating functional interactions among these factors in eosinophil progenitors. Our findings identify novel combinatorial protein-protein interactions for GATA-1, PU

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

Stankiewicz

Yang

et al. 2002

Journal of Biological Chemistry

Self Cite

106

139

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“…However, inducible Crk/CRKL complex formation and activation of Rap1 has been observed in di erent cell types and with several di erent stimuli (Ichiba et al, 1997;Garcia-Guzman et al, 1999;Alsayed et al, 2000;Du et al, 2000;Posern et al, 2000;Sakkab et al, 2000Sakkab et al, , 2001Xing et al, 2000). In some of these cases, for example in HGF-stimulated human embryonic kidney epithelial cells and Swiss 3T3 mouse ®broblasts stimulated with bombesin, it was clearly shown that Rap1 activation is dependent on Crk family adaptors Sakkab et al, 2000Sakkab et al, , 2001.…”

Section: C3gmentioning

confidence: 99%

Crk family adaptors–signalling complex formation and biological roles

2001

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T cell development and function in CrkL‐deficient mice

et al. 2003

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The adapter protein CrkL has been implicated in multiple signal transduction pathways in hematopoietic cells. In T lymphocytes, the recruitment of CrkL-C3G complexes has been correlated with hyporesponsiveness, implicating CrkL as a potential negative regulator. To test this hypothesis we examined T cell activation in CrkL-deficient mice. The CrkL -/-genotype was partially embryonic lethal. In viable CrkL -/-mice, peripheral blood counts were normal. The thymus from CrkL -/-mice had 40% fewer cells compared to littermates, but the proportion of thymocyte subsets was comparable. There was no discernable alteration in T cell function as reflected by T cell numbers, expression of memory markers, IL-2 production, proliferation, and differentiation into Th1/Th2 phenotypes. Immunization induced comparable levels of IgG2a and IgG1 antibodies. Chimeric mice, generated by transfer of CrkL -/-fetal liver cells into irradiated RAG2-/-recipients, also showed normal T cell function, arguing against selection via partial embryonic lethality. Our results indicate that CrkL is not absolutely required for T cell development or function, and argue against it being an essential component of a negative regulatory pathway in TCR signaling.

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Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils

Cited by 26 publications

References 84 publications

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

Crk family adaptors–signalling complex formation and biological roles

T cell development and function in CrkL‐deficient mice

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