Mechanism of p110γ-mediated growth suppression

磷酸肌肽3-激酶（PI3Ks）是一个进化上保守的脂质激酶家族，它调节大量的基本细胞反应，包括增殖、转化、分化和保护细胞凋亡。我们发现p110γ （PI3Kγ的催化亚基）的基因失活导致小鼠侵袭性结直肠癌的发展。在人类中，p110γ蛋白在患者的原发性结直肠癌和结肠癌细胞系中表达缺失。p110γ在人结肠癌细胞中的过表达导致其生长受到抑制。为了阐明p110γ抑制肿瘤作用的潜在机制，我们在体外研究了多种p110γ突变体抑制人类结直肠癌细胞系生长的能力。我发现HCT116和DLD1中p110γ激酶死亡突变体（R947P）的过表达抑制了细胞生长，通过集落形成实验和焦点实验进行了评估。这些结果表明p110γ抑制肿瘤的作用需要更多的K活性。由于生长抑制作用不依赖于它的酶活性，我们接下来检查哪些结构域结构是充分的和/或必要的效果。最近的结构研究表明，p110γ具有5个不同的结构域，即n端PH结构域、ras结合结构域、c2结构域、激酶结构域和螺旋结构域。ras结合结构域或激酶结构域的表达导致结肠癌细胞的生长抑制。然而，两种不能结合Ras的Ras结合突变体抑制细胞生长，野生型p110γ在缺乏内源性Ki-Ras表达的DLD1细胞系中表现出影响。此外，缺乏c -末端激酶结构域和完整ras结合结构域的截断突变体的表达对细胞生长没有影响。综上所述，这些结果表明，激酶结构域是p110γ抑制结肠癌细胞生长的必要和充分条件。值得注意的是，激酶活性对这些效应是必不可少的，这一结论为p110γ的功能提供了一个新的范式，并预测了抑制肿瘤发生的独特信号级联。少

Phosphoinositide 3-kinases (PI3Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. We found that genetic inactivation of p110γ, the catalytic subunit of PI3Kγ, leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110γ protein expression is lost in primary colorectal adenocarcinomas from patients and colon cancer ceil lines. Overexpression of p110γin human colon cancer cells resulted in suppression of their growth.To elucidate the underlying mechanism for the tumor suppressive effect of p110γ, we studied the ability of various p110γ mutants to suppress growth of human colorectal cancer cell lines in vitro. I found that overexpression of a kinase-dead mutant of p110γ (R947P) in HCT116 and DLD1 inhibited cell growth as assessed by colony formation assay and focus assay. These results suggest that P13 … More K activity is dispensable for the tumor suppressive effect of p110γ. As the growth suppressive effects did not depend on its enzymatic activity, we next examined which domain structure is sufficient and/or necessary for the effects. Recent structural studies have revealed that p110γpossesses five distinct domains, namely, N-terminal PH domain, Ras-binding domain, C2-domain, kinase domain and Helical domain. Expression of either the Ras-binding domain or the kinase domain led to growth suppression in the colon cancer cells. However, two Ras-binding mutants that cannot bind to Ras suppressed cell growth and wild type p110γ showed the effects in a DLD1 cell line that lacks endogenous Ki-Ras expression. In addition, expression of truncated mutants that lack the C-terminus kinase domain with intact Ras-binding domain had no effect on cell growth. Taken together, these results demonstrate that the kinase domain is necessary and sufficient for the growth suppression of the colon cancer cells by p110γ. Notably, the conclusion that the kinase activity is dispensable for the effects, provides a novel paradigm for p110γfunction and predict a unique signaling cascade that suppresses tumorigenesis. Less