Phospho-ULK1 (Ser555) (D1H4) R

5869:

Immunoprecipitation , Western Blotting

Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb detects endogenous levels of ULK1 only when phosphorylated at Ser555. Bands of unknown origin are detected between 90 and 100 kDa.

Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser555 of human ULK1 protein.

Western Blotting

Western blot analysis of extracts from MCF7 cells, untreated or treated with oligomycin #9996 (0.5 μM, 30 minutes), and C2C12 cells, untreated or treated with hydrogen peroxide (10 mM, 5 minutes), using Phospho-ULK1 (Ser555) (D1H4) Rabbit mAb.

Western Blotting

Western blot analysis of extracts from MCF7 cells, untreated or treated with oligomycin #9996 (0.5 µM, 30 minutes), using Phosho-ULK1 (Ser555) (D1H4) Rabbit mAb (left). Phoshpo-specificty is demonstrated by pre-incubating the antibody with phosphorylated (middle) or non-phoshporylated peptides (right) against a region surrounding Ser555 of ULK1.

Two related serine/threonine kinases, UNC-51-like kinase 1 and 2 (ULK1, ULK2), were discovered as mammalian homologs of the C. elegans gene UNC-51 in which mutants exhibited abnormal axonal extension and growth (1-4). Both proteins are widely expressed and contain an amino-terminal kinase domain followed by a central proline/serine rich domain and a highly conserved carboxy-terminal domain. The roles of ULK1 and ULK2 in axon growth have been linked to studies showing that the kinases are localized to neuronal growth cones and are involved in endocytosis of critical growth factors, such as NGF (5). Yeast two-hybrid studies found ULK1/2 associated with modulators of the endocytic pathway, SynGap and syntenin (6). Structural similarity of ULK1/2 has also been recognized with the yeast autophagy protein Atg1/Apg1 (7). Knockdown experiments using siRNA demonstrated that ULK1 is essential for autophagy (8), a catabolic process for the degradation of bulk cytoplasmic contents (9,10). It appears that Atg1/ULK1 can act as a convergence point for multiple signals that control autophagy (11), and can bind to several autophagy-related (Atg) proteins, regulating phosphorylation states and protein trafficking (12-16).

Phosphorylation of ULK1 by AMPK at Ser555 is critical for starvation-induced autophagy, cell survival under conditions of low nutrients and energy, and mitochondiral homeostasis (17).

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3. Yan, J. et al. (1998) Biochem Biophys Res Commun 246, 222-7.
4. Yan, J. et al. (1999) Oncogene 18, 5850-9.
5. Zhou, X. et al. (2007) Proc Natl Acad Sci USA 104, 5842-7.
6. Tomoda, T. et al. (2004) Genes Dev 18, 541-58.
7. Matsuura, A. et al. (1997) Gene 192, 245-50.
8. Chan, E.Y. et al. (2007) J Biol Chem 282, 25464-74.
9. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
10. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
11. Stephan, J.S. and Herman, P.K. (2006) Autophagy 2, 146-8.
12. Okazaki, N. et al. (2000) Brain Res Mol Brain Res 85, 1-12.
13. Young, A.R. et al. (2006) J Cell Sci 119, 3888-900.
14. Kamada, Y. et al. (2000) J Cell Biol 150, 1507-13.
15. Lee, S.B. et al. (2007) EMBO Rep 8, 360-5.
16. Hara, T. et al. (2008) J Cell Biol 181, 497-510.
17. Egan, D.F. et al. (2011) Science 331, 456-61.

• Wen, H. et al. (2011) Nat Immunol 12, 408-15. Applications: Western Blotting

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