Akt1 sequentially phosphorylates p27(kip1 )within a conserved but non-canonical region

BACKGROUND: p27(kip1 )(p27) is a multifunctional protein implicated in regulation of cell cycling, signal transduction, and adhesion. Its activity is controlled in part by Phosphatylinositol-3-Kinase (PI3K)/Akt1 signaling, and disruption of this regulatory connection has been identified in human breast cancers. The serine/threonine protein kinase Akt1 directly phosphorylates p27, so identifying the modified residue(s) is essential for understanding how it regulates p27 function. Various amino acids have been suggested as potential targets, but recent attention has focused on threonine 157 (T157) because it is located in a putative Akt1 consensus site. However, T157 is not evolutionarily conserved between mouse and human. We therefore re-evaluated Akt1 phosphorylation of p27 using purified proteins and in cells. RESULTS: Here we show purified Akt1 phosphorylates human and mouse p27 equally well. Phospho-peptide mapping indicates Akt1 targets multiple sites conserved in both species, while phospho-amino acid analysis identifies the targeted residues as serine rather than threonine. P27 deletion mutants localized these sites to the N-terminus, which contains the major p27 phosphorylation site in cells (serine 10). P27 phosphorylated by Akt1 was detected by a phospho-S10 specific antibody, confirming this serine was targeted. Akt1 failed to phosphorylate p27S10A despite evidence of a second site from mapping experiments. This surprising result suggested S10 phosphorylation might be required for targeting the second site. We tested this idea by replacing S10 with threonine, which as expected led to the appearance of phospho-threonine. Phospho-serine was still present, however, confirming Akt1 sequentially targets multiple serines in this region. We took two approaches in an attempt to explain why different residues were previously implicated. A kinetic analysis revealed a putative Akt1 binding site in the C-terminus, which may explain why mutations in this region affect p27 phosphorylation. Furthermore, commercially available recombinant Akt1 preparations exhibit striking differences in substrate specificity and site selectivity. To confirm S10 is a relevant site, we first showed that full-length wild type Akt1 purified from mammalian cells phosphorylates both human and mouse p27 on S10. Finally, we found that in cultured cells under physiologically relevant conditions such as oxidative stress or growth factor deprivation, endogenous Akt1 causes p27 accumulation by phosphorylating S10. CONCLUSION: Identifying where Akt1 phosphorylates p27 is essential for understanding its functional implications. We found that full-length wild type Akt1 – whether purified, transiently overexpressed in cells, or activated in response to cellular stress – phosphorylates p27 at S10, a noncanonical but evolutionarily conserved site known to regulate p27 activity and stability. Using recombinant Akt1 recapitulating this specificity, we showed modification of p27S10 also leads to phosphorylation of an adjacent serine. These results integrate PI3K/Akt1 signaling in response to stress with p27 regulation through its major phosphorylation site in cells, and thus identify new avenues for understanding p27 deregulation in human cancers

Tenfibgen Ligand Nanoencapsulation Delivers Bi-Functional Anti-CK2 RNAi Oligomer to Key Sites for Prostate Cancer Targeting Using Human Xenograft Tumors in Mice

<div><p>Protected and specific delivery of nucleic acids to malignant cells remains a highly desirable approach for cancer therapy. Here we present data on the physical and chemical characteristics, mechanism of action, and pilot therapeutic efficacy of a tenfibgen (TBG)-shell nanocapsule technology for tumor-directed delivery of single stranded DNA/RNA chimeric oligomers targeting CK2αα' to xenograft tumors in mice. The sub-50 nm size TBG nanocapsule (s50-TBG) is a slightly negatively charged, uniform particle of 15 - 20 nm size which confers protection to the nucleic acid cargo. The DNA/RNA chimeric oligomer (RNAi-CK2) functions to decrease CK2αα' expression levels via both siRNA and antisense mechanisms. Systemic delivery of s50-TBG-RNAi-CK2 specifically targets malignant cells, including tumor cells in bone, and at low doses reduces size and CK2-related signals in orthotopic primary and metastatic xenograft prostate cancer tumors. In conclusion, the s50-TBG nanoencapsulation technology together with the chimeric oligomer targeting CK2αα' offer significant promise for systemic treatment of prostate malignancy.</p></div

Nanocapsule design, morphology, cargo stability, and cargo protection.

<p>(<b>a</b>) Cartoon depiction of nanocapsule design. (<b>b</b>) Transmission electron micrograph of s50-TBG-RNAi-CK2 nanocapsules for <i>in vivo</i> studies. Magnification 230,000×. Scale bar 100 nm. (<b>c</b>) Left panel: Naked RNAi-CK2 oligomer was digested with proteinase K for 24 to 96 h. Inp, undigested input oligomer. Right panel: Naked and s50-TBG encapsulated RNAi-CK2 oligomers were digested with DNase followed by proteinase K as indicated above the panel. Lanes 1 & 2, naked RNAi-CK2; 3 & 4, naked RNAi-CK2 with TBG-sugar nanocapsules included in the digestion; 5 - 7, <i>in vitro</i> use formulation of s50-TBG-RNAi-CK2; 8 – 10, <i>in vivo</i> use formulation of s50-TBG-RNAi-CK2.</p

Cellular uptake of s50-TBG nanocapsules and effects of s50-TBG-RNAi-CK2 in benign and malignant prostate cells.

<p>(<b>a</b>) Uptake over 24 h of s50-TBG nanocapsules with FeO-dextran cargo in PC3-LN4 cells plated onto TnFn-3D. Cells were stained with DAB-enhanced Prussian blue for iron and counterstained with Fast Red. Scale bar 100 µm. (<b>b</b>) Malignant cell-specific uptake of s50-TBG nanocapsules. s50-TBG-FeO-dextran uptake was determined by iron staining at 8 h in PC3-LN4 and BPH-1 cells grown on TnFn- or laminin-coated nanofiber scaffolds, respectively. Scale bar 100 µm. (<b>c</b>) Cellular proliferation effects of s50-TBG-RNAi-CK2 treatment in benign and malignant prostate cells. PC3-LN4 and C4-2 grown on TnFn-3D and BPH-1 cells grown on laminin-3D in 96-well plates were treated with s50-TBG-RNAi-CK2 or control TBG nanocapsules containing RNAi-RFP-6R targeting Red Fluorescence Protein as indicated. ³H-thymidine was added after 48 h, and cells analyzed at 72 h post-nanocapsule addition. Results are expressed relative to treatment with s50-TBG-sugar nanocapsules. The s50-TBG nanocapsule cargo and cell lines used are indicated below the bars. Means ± SE are presented (n = 3 for all). *p<0.005 relative to s50-TBG-sugar and –RFP; # p<0.01 relative to TBG-sugar; $ p = 0.006 relative to TBG-RFP. (<b>d</b>) s50-TBG-RNAi-CK2 treatment reduced CK2α and CK2α' mRNA steady-state expression levels in PC3-LN4 cells. mRNA isolated from PC3-LN4 cells grown on TnFn 24 and 48 h after s50-TBG-RNAi-CK2 or –sugar treatment as indicated was analyzed by reverse transcriptase real-time quantitative PCR for CK2α and CK2α' expression. HPRT transcript was used to normalize expression levels. Means, SE and p-values are presented (24 h CK2α n = 5, CK2α' n = 6; 48 h CK2α n = 2, CK2α' n = 2).</p