Caveolin-1 Maintains Activated Akt in Prostate CancerCells through Scaffolding Domain Binding Site Interactions with andInhibition of Serine/Threonine Protein Phosphatases PP1 andPP2A

Abstract

Previously it has been reported that caveolin-1 (cav-1) has antiapoptotic activities in prostate cancer cells and functions downstream of androgenic stimulation. In this study, we demonstrate that cav-1 overexpression significantly reduced thapsigargin (Tg)-stimulated apoptosis. Examination of the phosphatidylinositol 3-kinase (PI3-K)/Akt signaling cascade revealed higher activities of PDK1 and Akt but not PI3-K in cav-1-stimulated cells compared to control cells. We subsequently found that cav-1 interacts with and inhibits serine/threonine protein phosphatases PP1 and PP2A through scaffolding domain binding site interactions. Deletion of the cav-1 scaffolding domain significantly reduces phosphorylated Akt and cell viability compared with wild-type cav-1. Analysis of potential substrates for PP1 and PP2A revealed that cav-1-mediated inhibition of PP1 and PP2A leads to increased PDK1, Akt, and ERK1/2 activities. We demonstrate that increased Akt activities are largely responsible for cav-1-mediated cell survival using dominant-negative Akt mutants and specific inhibitors to MEK1/MEK and show that cav-1 increases the half-life of phosphorylated PDK1 and Akt after inhibition of PI3-K by LY294002. We further demonstrate that cav-1-stimulated Akt activities lead to increased phosphorylation of multiple Akt substrates, including GSK3, FKHR, and MDM2. In addition, overexpression of cav-1 significantly increases translocation of phosphorylated androgen receptor to nucleus. Our studies therefore reveal a novel mechanism of Akt activation in prostate cancer and potentially other malignancies.Prostate cancer remains the second leading cause of cancer mortality among American males. The predominant reason for such high and persistent mortality is the lack of curative therapies for androgen-resistant metastatic disease. It is critical to elucidate the molecular mechanisms that underlie the ultimate androgen-resistant state of prostate cancer and to develop effective therapies for this condition.Previously, Yang et al. reported that caveolin-1 (cav-1) levels were elevated in metastatic mouse and human prostate cancer (85). cav-1 is a major component of caveolae, flask-shaped membrane invaginations which are involved in multiple cellular processes, including the regulation and transportation of cellular cholesterol and lipids, clathrin-independent endocytosis, and signal transduction (24,27,60,62,66). The participation of cav-1 in these critical pathways involves the interaction of cav-1 with a relatively large number of molecules in either a scaffolding binding-dependent or -independent manner (41, 63). The wide spectrum of molecular interactions involving cav-1 is consistent with important, context-dependent roles for cav-1 in signal transduction, molecular transport, and other regulatory activities.The biological functions of cav-1 in cancer are complex, multifaceted, and somewhat controversial (42,55,72,73). Numerous experimental results indicate that cav-1 is a growth suppressor (14,17,35). Some investigators have assert...

The emerging role of the PI3-K-Akt pathway in prostate cancer progression

“…This PI3-Kindependent Akt activation in cav-1 expressing cells was further confirmed by the determination of P-Akt half-life using the PI3-K-specific inhibitor LY294002. 41 These data suggested that cav-1 participates in the regulation of Akt by maintaining Akt in the activated (phosphorylated) form.…”

“…57 Importantly, our studies in prostate cancer cells showed that cav-1-mediated maintenance of activated Akt leads to increased phosphorylation of multiple downstream targets, including GSK3a/b, FKHRL, and MDM2. 41 All of these three proteins play important roles in cancer cell survival as discussed above.…”

“…Interestingly, the catalytic subunits of PP1 and PP2A possess a previously described consensus cav-1 binding site. 42 Immunoprecipitation (IP) experiments demonstrated that cav-1 was present in the PP1 or Akt pathway in prostate cancer L Li et al PP2A IP complex and PP1 or PP2A was also present in the cav-1 IP complex, 41 suggesting that cav-1 associates with PP1 and PP2A. In vitro IP experiments using purified His-V5-human cav-1, PP1 (catalytic subunit) and PP2A (core enzyme) further demonstrated that cav-1 could bind to PP1 and PP2A outside of an intact cell context.…”

“…Importantly, serine/threonine protein phosphatase activity assays using PP1 or PP2A IP complex or purified recombinant enzyme (PP1 catalytic subunit or PP2A core enzyme) indicated that interaction with cav-1 leads to the inhibition of PP1 and PP2A activities. 41 Reduced overall activities of PP1 and PP2A favor the maintenance of Akt phosphorylation and increased Akt activities.…”

“…122 On the other hand, the effectors that regulate and activate Akt activity may be also 'drugable'. For example, the elevated cav-1 in prostate cancer favors the maintenance of activated Akt, 41 and therefore targeting cav-1 provides an alternative approach to suppressing Akt activity in prostate cancer cells. Since cav-1 is secreted by prostate cancer cells and can be detected in the serum of prostate cancer patients, 123,124 using a specific antibody to neutralize cav-1 may be therapeutically useful.…”