The COOH-Terminal Domain of Wild-Type Cot Regulates Its Stability and Kinase Specific Activity

Abstract

Cot, initially identified as an oncogene in a truncated form, is a mitogen-activated protein kinase kinase kinase implicated in cellular activation and proliferation. Here, we show that this truncation of Cot results in a 10-fold increase in its overall kinase activity through two different mechanisms. Truncated Cot protein exhibits a lower turnover rate (half-life, 95 min) than wild-type Cot (half-life, 35 min). The degradation of wild-type and truncated Cot can be specifically inhibited by proteasome inhibitors in situ. The 20S proteasome also degrades wild-type Cot more efficiently than the truncated protein. Furthermore, the amino acid 435 to 457 region within the wild-type Cot COOH-terminal domain confers instability when transferred to the yellow fluorescent protein and targets this fusion protein to degradation via the proteasome. On the other hand, the kinase specific activity of wild-type Cot is 3.8-fold lower than that of truncated Cot, and it appears that the last 43 amino acids of the wild-type Cot COOH-terminal domain are those responsible for this inhibition of kinase activity. In conclusion, these data demonstrate that the oncogenic activity of truncated Cot is the result of its prolonged half-life and its higher kinase specific activity with respect to wild-type Cot.The Cot/tpl-2 gene encodes a mitogen-activated protein (MAP) kinase kinase kinase that is potentially capable of switching on several MAP kinase cascades, namely those leading to the activation of the MAP kinases ERK1/ERK2, JNK, p38␥, and ERK5 (13,20,25,38,47,53). These signal transduction pathways link Cot activity with the up-regulation of several transcription factors, such as AP

Regulation and function of TPL-2, an IκB kinase-regulated MAP kinase kinase kinase

“…Steady-state levels of TPL-2 are very low in p105-deficient Nfκb1 −/− cells [34,38], and consequently LPS activation of MEK and ERK is substantially reduced in bone-marrow-derived macrophages (BMDM) generated from Nfκb1 −/− mice [34]. Since the proposed degron sequence of TPL-2 is located within one of the binding sites for p105, it is possible that this interaction stabilizes TPL-2 by covering the degron [8]. Second, direct interaction of p105 with the kinase domain of TPL-2 inhibits TPL-2 MEK kinase activity by preventing access to MEK [34,38].…”

“…C-terminal truncation, however, results in a protein (TPL-2ΔC) with increased kinase-specific activity, suggesting that this region may inhibit TPL-2 kinase activity [6]. Furthermore, a proposed degron sequence (435-457, shaded box) is located within the C terminus and confers destabilizing properties to full-length TPL-2 [8]. Consequently, TPL-2∆C has increased protein stability and is expressed at higher levels.…”

“…First, C-terminal truncation increases the specific kinase activity of TPL-2, and it has been suggested that the C terminus may modulate TPL-2 catalytic activity by folding back onto the kinase domain [6,8]. Second, C-terminal truncation removes a degron sequence (amino acids 435-457) that promotes proteolysis of TPL-2 by the proteasome [8]. Consequently, TPL-2ΔC is expressed at higher levels in cells than TPL-2.…”

Cot/tpl2 activity is required for TLR‐induced activation of the Akt p70 S6k pathway in macrophages: Implications for NO synthase 2 expression

“…The partial proteolysis of p105 NFkB to p50 NF-kB releases Cot/tpl-2 from the complex [15][16][17][18][19][20]. Dissociated and adequately phosphorylated Cot/tpl2 [21][22][23][24] fully activates MKK1 and consequently Erk1/2, prior to being rapidly degraded through the proteasome pathway [16,17,[25][26][27]. Cot/tpl-2 (MAP3K8) is the sole MAP3K that activates the MKK1-Erk1/2 pathway in response to the activation of receptors of the TLR/IL-1 superfamily, as well as some of the TNF receptor family [17,[28][29][30][31][32][33][34].…”

“…The partial proteolysis of p105 NFkB to p50 NF-kB releases Cot/tpl-2 from the complex [15][16][17][18][19][20]. Dissociated and adequately phosphorylated Cot/tpl2 [21][22][23][24] fully activates MKK1 and consequently Erk1/2, prior to being rapidly degraded through the proteasome pathway [16,17,[25][26][27].…”