Twist1, a basic helix-loop-helix transcription factor, promotes breast tumor 
cell epithelial-mesenchymal transition (EMT), invasiveness, and metastasis. 
However, the mechanisms responsible for regulating Twist1 stability are unknown 
in these cells. We identified the serine 68 (Ser 68) as a major phosphorylation 
site of Twist1 by mass spectrometry and with specific antibodies. This Ser 68 is 
phosphorylated by p38, c-Jun N-terminal kinases (JNK), and extracellular 
signal-regulated kinases1/2 in vitro, and its phosphorylation levels positively 
correlate with Twist1 protein levels in human embryonic kidney 293 and breast 
cancer cells. Prevention of Ser 68 phosphorylation by an alanine (A) mutation 
(Ser 68A) dramatically accelerates Twist1 ubiquitination and degradation. 
Furthermore, activation of mitogen-activated protein kinases (MAPK) by an active 
Ras protein or TGF-β treatment significantly increases Ser 68 phosphorylation 
and Twist1 protein levels without altering Twist1 mRNA expression, whereas 
blocking of MAPK activities by either specific inhibitors or dominant negative 
inhibitory mutants effectively reduces the levels of both induced and uninduced 
Ser 68 phosphorylation and Twist protein. Accordingly, the mammary epithelial 
cells expressing Twist1 exhibit much higher degrees of EMT and invasiveness on 
stimulation with TGF-β or the active Ras and paclitaxel resistance compared with 
the same cells expressing the Ser 68A-Twist1 mutant. Importantly, the levels of 
Ser 68 phosphorylation in the invasive human breast ductal carcinomas positively 
correlate with the levels of Twist1 protein and JNK activity and are 
significantly higher in progesterone receptor-negative and HER2-positive breast 
cancers. These findings suggest that activation of MAPKs by tyrosine kinase 
receptors and Ras signaling pathways may substantially promote breast tumor cell 
EMT and metastasis via phoshorylation and stabilization of Twist1.