Mechanism Study On ULK1-mediated Phosphorylation Of Exo70 And The Consequent Inhibition Of Breast Cancer Cell Metastasis

Distant metastasis and local recurrence are the main causes of death in patients with breast cancer,so it is of great significance to study the regulatory mechanisms of breast cancer metastasis.Exo70,a key subunit of Exocyst complex,plays important roles in the regulation of tumor cell migration and invasion via promoting the formation of pseudopodia and secretion of matrix metalloproteinases.However,there are few reports about the modulation of Exo70.Expression level of serine/threonine protein kinase ULK1 was negatively correlated with the lymph node metastasis in breast cancer,suggesting that ULK1 may be involved in the regulation of breast cancer metastasis,but its exact role and the underlying molecular mechanism remain unclear.In this study,we found that the expression levels of endogenous ULK1 in several human breast cancer cell lines were negatively correlated with their metastatic potential.Overexpression of ULK1 significantly inhibited the migration and invasion ability of highly metastatic breast cancer cells,and the kinase activity of ULK1 was required;on the other hand,knocking down endogenous ULK1 in low metastatic breast cancer cells could remarkably increase their migration and invasion capacity.Further mechanistic study identified a novel substrate of kinase ULK1,i.e.,tumor metastasis-associated protein Exo70.ULK1 and Exo70 were co-localized in breast cancer cells,and ULK1 directly bound to and phosphorylated Exo70.We further found that Exo70 was essential for ULK1 to suppress the migration and invasion of breast cancer cells,suggesting that the inhibitory effect of ULK1 on breast cancer metastasis might be through phosphorylating Exo70.By tandem mass spectrometry,conservative sequence alignment of ULK1 substrates,inactivating mutations of phosphorylation sites,and cellular and in vitro phosphorylation experiments,we determined that Exo70 was phosphorylated at residues Ser47,Ser59 and Ser89 by ULK1.Furthermore,cellular and mouse model experiments with constitutive activating mutations of these three phosphorylation sites indicated that phosphorylation of Exo70 protein at Ser47,Ser59 and Ser89 by ULK1 greatly weakened the oligomerization of Exo70 and Exocyst formation,leading to reduced pseudopodia formation and exocytosis secretion,and thereby suppressed the migration and invasion of breast cancer cells.In summary,for the rst time,our present study demonstrated the role and the underlying molecular mechanisms of protein kinase ULK1 to suppress breast cancer metastasis,namely through direct binding and phosphorylating its new substrate Exo70,which weakened the oligomerization of Exo70 and Exocyst formation then subsequenly decreased pseudopodia formation and exocytosis secretion,resulting in the inhibition of breast cancer metastasis.