Pak regulates calpain-dependent degradation of E3b1.
E3b1, a binding partner of Eps8, plays a critical role in receptor tyrosine kinase (RTK)-mediated Rac activation by facilitating the interaction of Eps8 with Sos-1 and the consequent activation of the Rac-specific guanine nucleotide exchange factor activity of Sos-1. Here we present evidence that E3b1 levels are regulated by the Ca(2+)-activated protease calpain, and also by Pak, a downstream target of Rac signaling. Serum starvation of Rat2 or COS7 cells resulted in rapid loss of E3b1 that was reversed by calpain inhibitors. Loss was also prevented by expressing the constitutively active Pak1 mutant, Pak1(H83,86L). Activation of endogenous Pak by platelet-derived growth factor or the constitutively active Rac1 mutant, Rac1(G12V), also inhibited degradation. In contrast, inhibition of endogenous Pak activity by expressing the Pak auto-inhibitory domain caused degradation of over-expressed E3b1 even in the presence of serum. Taken together, these findings indicate that E3b1 is down-regulated by calpain activation and stabilized by Pak activation. They also suggest that RTK-mediated Rac activation can be modulated by changes in the level of E3b1 in response to signals that affect the activity of calpain or Pak.
Chi
Susan
S
School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Chang
Soohee
S
Park
Dongeun
D
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D017931:DNA Primers; D004789:Enzyme Activation; D006868:Hydrolysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D054462:p21-Activated Kinases
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D017931:DNA Primers; C515906:Pak1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D002154:Calpain