Mouse ULK2, a novel member of the UNC-51-like protein kinases: unique features of functional domains

Abstract

The UNC-51 serine/threonine kinase of C. elegans plays an essential role in axonal elongation, and unc-51 mutants exhibit uncoordinated movements. We have previously identi®ed mouse and human cDNAs encoding UNC-51-like kinase (ULK1). Here we report the identi®cation and characterization of the second murine member of this kinase family, ULK2. Mouse ULK2 cDNA encodes a putative polypeptide of 1033 aa which has an overall 52% and 33% amino acid identity to ULK1 and UNC-51, respectively. ULKs and UNC-51 share a typical domain structure of an amino-terminal kinase domain, a central proline/serine rich (PS) domain, and a carboxy-terminal (C) domain. Northern blot analysis showed that ULK2 mRNA is widely expressed in adult tissues. In situ hybridization analysis indicated that ULK2 mRNA is ubiquitously localized in premature as well as mature neurons in developing nervous system. ULK2 gene was mapped to mouse chromosome 11B1.3 and rat chromosome 10q23 by FISH. HA-tagged ULK2 expressed in COS7 cells had an apparent molecular size of *150 kDa and was autophosphorylated in vitro. Truncation mutants suggested that the autophosphorylation occurs in the PS domain. Although expression of ULK2 failed to rescue unc-51 mutant of C. elegans, a series of ULK2/UNC-51 chimeric kinases revealed that function of the kinase and PS domains are conserved among species, while the C domain acts in a speciesspeci®c manner. These results suggest that ULK2 is involved in a previously uncharacterized signaling pathway in mammalian cells.

siRNA Screening of the Kinome Identifies ULK1 as a Multidomain Modulator of Autophagy

“…Unlike the previously described model systems, vertebrates contain two genes, ULK1 and ULK2, that encode proteins with close homology to Atg1p. Both ULK1 and ULK2 are widely expressed in mouse tissues and kinase-dead versions of ULK1 and ULK2 can act as dominant negative proteins in neurite outgrowth assays (34,38). We found that knockdown of ULK2 did not inhibit GFP-LC3 conversion, suggesting that only ULK1 has autophagy regulatory roles, at least in 293 cells.…”

“…Studies performed using Dictyostelium, Drosophila, and C. elegans have confirmed a role for their Atg1 homologs in autophagy (6,7,31,32). In mammals, two Atg1 homologs have been identified, uncoordinated 51-like kinase 1 (ULK1) and ULK2 (33,34), and we recently demonstrated an involvement of ULK1 in the subcellular re-distribution of mammalian Atg9 that takes place following nutrient starvation (35). Evidence from Drosophila and mammalian cells also indicate that Atg1 proteins have the ability to signal "upstream" to TOR via a feedback loop (32,36).…”

Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies

“…2 Atg1 homologues have been reported in several species, including mammals, where there are two Atg1 homologues, UNC (uncoordinated movement)-51-like kinase 1 (ULK1) and ULK2. 76,77 Tooze and coworkers 78 have shown that siRNA-mediated knockdown of ULK1, but not ULK2, inhibited rapamycin-induced autophagy, further suggesting a role downstream of mTOR. This is consistent with our earlier finding in which rapamycin failed to rescue neurodegeneration in Drosophila with heterozygous loss of Atg1.…”

A novel, human Atg13 binding protein, Atg101, interacts with ULK1 and is essential for macroautophagy

“…9,14 The autophagy process and many of the autophagy-specific proteins are conserved in higher eukaryotes. Unc-51 like kinase 1 (ULK1) and unc-51 like kinase 2 (ULK2) have sequence homology to Atg1, [15][16][17] but only recently was ULK1 confirmed to have a functional role in autophagy. Three different studies have used siRNA to deplete cells of ULK1, demonstrating that ULK1 is important for the starvation-induced trafficking of mAtg9, for lipid modification of GFP-LC3, and for the macroautophagymediated degradation of a cytosolic protein.…”