Directional sensing requires G beta gamma-mediated PAK1 and PIX alpha-dependent activation of Cdc42

Efficient chemotaxis requires directional sensing and cell polarization. We describe a signaling mechanism involving G beta gamma, PAK-associated guanine nucleotide exchange factor (PIX alpha), Cdc42, and p21-activated kinase (PAK) 1. This pathway is utilized by chemoattractants to regulate directional sensing and directional migration of myeloid cells. Our results suggest that G beta gamma binds PAK1 and, via PAK-associated PIX alpha, activates Cdc42, which in turn activates PAK1. Thus, in this pathway, PAK1 is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation. This G beta gamma-PAK1/PIX alpha/Cdc42 pathway is essential for the localization of F-actin formation to the leading edge, the exclusion of PTEN from the leading edge, directional sensing, and the persistent directional migration of chemotactic leukocytes. Although ligand-induced production of PIP(3) is not required for activation of this pathway, PIP(3) appears to localize the activation of Cdc42 by the pathway.     

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.     

Phosphoinositides and Rho proteins spatially regulate actin polymerization to initiate and maintain directed movement in a one-dimensional model of a motile cell

Gradient sensing, polarization, and chemotaxis of motile cells involve the actin cytoskeleton, and regulatory modules, including the phosphoinositides (PIs), their kinases/phosphatases, and small GTPases (Rho proteins). Here we model their individual components (PIP1, PIP2, PIP3; PTEN, PI3K, PI5K; Cdc42, Rac, Rho; Arp2/3, and actin), their interconversions, interactions, and modular functions in the context of a one-dimensional dynamic model for protrusive cell motility, with parameter values derived from in vitro and in vivo studies. In response to a spatially graded stimulus, the model produces stable amplified internal profiles of regulatory components, and initiates persistent motility (consistent with experimental observations). By connecting the modules, we find that Rho GTPases work as a spatial switch, and that the PIs filter noise, and define the front versus back. Relatively fast PI diffusion also leads to selection of a unique pattern of Rho distribution from a collection of possible patterns. We use the model to explore the importance of specific hypothesized interactions, to explore mutant phenotypes, and to study the role of actin polymerization in the maintenance of the PI asymmetry. We also suggest a mechanism to explain the spatial exclusion of Cdc42 and PTEN and the inability of cells lacking active Cdc42 to properly detect chemoattractant gradients.     

Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration

Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.     

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.     

Co-existence of high levels of the PTEN protein with enhanced Akt activation in renal cell carcinoma

Recruiting Akt to the membrane-bound phosphatidylinositol (3,4,5) trisphosphate (PIP3) is required for Akt activation. While PI3 kinase (PI3K) produces PIP3, PTEN dephosphorylates the 3-position phosphate from PIP3, thereby directly inhibiting Akt activation. PTEN is the dominant PIP3 phosphatase, as knockdown of PTEN results in increases in Akt activation in mice. The PTEN tumor suppressor gene is frequently mutated in a variety of human cancers, consistent with an inverse correlation between levels of the PTEN protein and Akt activation. We have examined PTEN expression and Akt activation in 35 primary clear cell renal cell carcinomas RCCs (ccRCCs) and 9 papillary RCCs (pRCCs) and their respective non-tumor kidney tissues. The PTEN protein was reduced in 16 ccRCCs (16/35=45.7%) and 8 pRCCs (8/9=88.9%). In these RCCs, 25.0% (4/16) of ccRCCs and 25.0% (2/8) of pRCCs expressed elevated Akt activation. 19 ccRCCc (19/35=54.3%) expressed comparable or higher levels of PTEN. Of these ccRCCs, 31.6% (6/19) showed increases in Akt activation. As PTEN dominantly inhibits Akt activation, the coexistence of high levels of the PTEN protein with enhanced Akt activation suggests the existence of novel mechanisms which attenuate PTEN function in ccRCC. These mechanisms may reduce PTEN function or increase PIP3 production.     

PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway.

The tumor suppressor PTEN is a phosphatase with sequence homology to tensin. PTEN dephosphorylates phosphatidylinositol 3,4, 5-trisphosphate (PIP3) and focal adhesion kinase (FAK), and it can inhibit cell growth, invasion, migration, and focal adhesions. We investigated molecular interactions of PTEN and FAK in glioblastoma and breast cancer cells lacking PTEN. The PTEN trapping mutant D92A bound wild-type FAK, requiring FAK autophosphorylation site Tyr397. In PTEN-mutated cancer cells, FAK phosphorylation was retained even in suspension after detachment from extracellular matrix, accompanied by enhanced PI 3-K association with FAK and sustained PI 3-K activity, PIP3 levels, and Akt phosphorylation; expression of exogenous PTEN suppressed all five properties. PTEN-mutated cells were resistant to apoptosis in suspension, but most of the cells entered apoptosis after expression of exogenous PTEN or wortmannin treatment. Moreover, overexpression of FAK in PTEN-transfected cells reversed the decreased FAK phosphorylation and PI 3-K activity, and it partially rescued PIP3 levels, Akt phosphorylation, and PTEN-induced apoptosis. Our results show that FAK Tyr397 is important in PTEN interactions with FAK, that PTEN regulates FAK phosphorylation and molecular associations after detachment from matrix, and that PTEN negatively regulates the extracellular matrix-dependent PI 3-K/Akt cell survival pathway in a process that can include FAK.     

Tumor suppressor PTEN mediates sensing of chemoattractant gradients

Shallow gradients of chemoattractants, sensed by G protein-linked signaling pathways, elicit localized binding of PH domains specific for PI(3,4,5)P3 at sites on the membrane where rearrangements of the cytoskeleton and pseudopod extension occur. Disruption of the PI 3-phosphatase, PTEN, in Dictyostelium discoideum dramatically prolonged and broadened the PH domain relocation and actin polymerization responses, causing the cells lacking PTEN to follow a circuitous route toward the attractant. Exogenously expressed PTEN-GFP localized to the surface membrane at the rear of the cell. Membrane localization required a putative PI(4,5)P2 binding motif and was required for chemotaxis. These results suggest that specific phosphoinositides direct actin polymerization to the cell's leading edge and regulation of PTEN through a feedback loop plays a critical role in gradient sensing and directional migration.     

NHERFs, NEP, MAGUKs, and more: interactions that regulate PTEN

This year marks the 10th anniversary of the discovery of the PTEN/MMAC1/TEP1 tumor suppressor gene (hereafter referred to as PTEN), one of the most commonly mutated genes in cancer. PTEN encodes a lipid phosphatase that dephosphorylates phosphoinositide-3,4,5-triphosphate (PIP(3)), thereby counteracting mitogenic signaling pathways driven by phosphoinositol-3-kinases (PI3K). By opposing PI3K signaling, PTEN inhibits the activation of the critical PI3K effector proteins Akt1-3 (also known as protein kinase B or PKB). Given its central role in antagonizing PI3K signaling, one might expect that like PI3K, the activity of the PTEN protein would be highly regulated by numerous protein/protein interactions. However, surprisingly little is known about such interactions. This fact, combined with the generally accepted notion that phosphatases are less exquisitely regulated than kinases, has led to the idea that PTEN may function in a relatively unregulated fashion. Here we review the identities and proposed functions of known PTEN-interacting proteins, and point out avenues of investigation that we hope may be fruitful in identifying important new mechanisms of PTEN regulation in mammalian cells.     

Phosphatase PTEN is inactivated in bovine aortic endothelial cells exposed to cyclic strain

Hemodynamic forces, including cyclic strain (CS) and shear stress (SS), have been recognized as important modulators of vascular cell morphology and function. PTEN (also known as MMAC1/TEP1) is a lipid phosphatase that leads to a decrease in intracellular phosphatidylinositol 3,4,5 trisphosphate (PIP3) and therefore can modulate the stimulating effect of phosphatidylinositol 3-kinase (PI3K). In this study, we focused on the upstream regulators of the PI3K-Akt pathway by assessing Akt, PTEN, casein kinase 2 (CK2) (a kinase that catalyzes phosphorylation of PTEN), and PI3K activity in endothelial cells (EC) exposed to CS. The activity of phospho-PTEN (n = 4) and phospho-CK2 (n = 4) increased in a time-dependent fashion, reaching maximal activity by 10 min of CS stimulation. The peak of phospho-Akt activity (n = 4) occurred later, at 60 min. Akt activity was altered by transfection of EC with dominant negative PTEN plasmids. Furthermore, CS increased PIP3 immunoreactivity in a time-dependent manner, reaching maximal activity after 60 min of CS stimulation, and these effects were affected by transfection of EC with dominant negative PTEN plasmids. Inhibition of PTEN activity had no effect on CS-mediated cell proliferation but inhibited CS-mediated suppression of apoptosis.     

PTEN expression causes feedback upregulation of insulin receptor substrate 2

PTEN is a tumor suppressor that antagonizes phosphatidylinositol-3 kinase (PI3K) by dephosphorylating the D3 position of phosphatidylinositol (3,4,5)-triphosphate (PtdIns-3,4,5-P3). Given the importance of PTEN in regulating PtdIns-3,4,5-P3 levels, we used Affymetrix GeneChip arrays to identify genes regulated by PTEN. PTEN expression rapidly reduced the activity of Akt, which was followed by a G(1) arrest and eventually apoptosis. The gene encoding insulin receptor substrate 2 (IRS-2), a mediator of insulin signaling, was found to be the most induced gene at all time points. A PI3K-specific inhibitor, LY294002, also upregulated IRS-2, providing evidence that it was the suppression of the PI3K pathway that was responsible for the message upregulation. In addition, PTEN, LY294002, and rapamycin, an inhibitor of mammalian target of rapamycin, caused a reduction in the molecular weight of IRS-2 and an increase in the association of IRS-2 with PI3K. Apparently, PTEN inhibits a negative regulator of IRS-2 to upregulate the IRS-2-PI3K interaction. These studies suggest that PtdIns-3,4,5-P3 levels regulate the specific activity and amount of IRS-2 available for insulin signaling.     

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2)

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.     

Basal B cell receptor-directed phosphatidylinositol 3-kinase signaling turns off RAGs and promotes B cell-positive selection

PI3K plays key roles in cell growth, differentiation, and survival by generating the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3). PIP3 activates numerous enzymes, in part by recruiting them from the cytosol to the plasma membrane. We find that in immature B lymphocytes carrying a nonautoreactive Ag receptor, PI3K signaling suppresses RAG expression and promotes developmental progression. Inhibitors of PI3K signaling abrogate this positive selection. Furthermore, immature primary B cells from mice lacking the p85alpha regulatory subunit of PI3K suppress poorly RAG expression, undergo an exaggerated receptor editing response, and, as in BCR-ligated cells, fail to progress into the G1 phase of cell cycle. Moreover, immature B cells carrying an innocuous receptor have sustained elevation of PIP3 levels and activation of the downstream effectors phospholipase C (PLC)gamma2, Akt, and Bruton's tyrosine kinase. Of these, PLCgamma2 appears to play the most significant role in down-regulating RAG expression. It therefore appears that when the BCR of an immature B cell is ligated, PIP3 levels are reduced, PLCgamma2 activation is diminished, and receptor editing is promoted by sustained RAG expression. Taken together, our results provide evidence that PI3K signaling is an important cue required for fostering development of B cells carrying a useful BCR.     

Oxidative inactivation of the lipid phosphatase phosphatase and tensin homolog on chromosome ten (PTEN) as a novel mechanism of acquired long QT syndrome

The most common cause of cardiac side effects of pharmaco-therapy is acquired long QT syndrome, which is characterized by abnormal cardiac repolarization and most often caused by direct blockade of the cardiac potassium channel human ether a-go-go-related gene (hERG). However, little is known about therapeutic compounds that target ion channels other than hERG. We have discovered that arsenic trioxide (As(2)O(3)), a very potent antineoplastic compound for the treatment of acute promyelocytic leukemia, is proarrhythmic via two separate mechanisms: a well characterized inhibition of hERG/I(Kr) trafficking and a poorly understood increase of cardiac calcium currents. We have analyzed the latter mechanism in the present study using biochemical and electrophysiological methods. We find that oxidative inactivation of the lipid phosphatase PTEN by As(2)O(3) enhances cardiac calcium currents in the therapeutic concentration range via a PI3Kα-dependent increase in phosphatidylinositol 3,4,5-triphosphate (PIP(3)) production. In guinea pig ventricular myocytes, even a modest reduction in PTEN activity is sufficient to increase cellular PIP(3) levels. Under control conditions, PIP(3) levels are kept low by PTEN and do not affect calcium current amplitudes. Based on pharmacological experiments and intracellular infusion of PIP(3), we propose that in guinea pig ventricular myocytes, PIP(3) regulates calcium currents independently of the protein kinase Akt along a pathway that includes a secondary oxidation-sensitive target. Overall, our report describes a novel form of acquired long QT syndrome where the target modified by As(2)O(3) is an intracellular signaling cascade.     

Role of PI3-kinase and PI4-kinase in actin polymerization during bovine sperm capacitation

We have recently demonstrated the involvement of phospholipase D (PLD) in actin polymerization during mammalian sperm capacitation. In the present study, we investigated the involvement of phosphatidylinositol 3- and 4-kinases (PI3K and PI4K) in actin polymerization, as well as the production of PIP(2(4,5)), which is a known cofactor for PLD activation, during bovine sperm capacitation. PIK3R1 (p85 alpha regulatory subunit of PI3K) and PIKCB (PI4K beta) in bovine sperm were detected by Western blotting and immunocytochemistry. Wortmannin (WT) inhibited PI3K and PI4K type III at concentrations of 10 nM and 10 microM, respectively. PI4K activity and PIP(2(4,5)) production were blocked by 10 microM WT but not by 10 nM WT, whereas PI3K activity and PIP(3(3,4,5)) production were blocked by 10 nM WT. Moreover, spermine, which is a known PI4K activator and a component of semen, activated sperm PI4K, resulting in increased cellular PIP(2(4,5)) and F-actin formation. The increases in PIP(2(4,5)) and F-actin intracellular levels during sperm capacitation were mediated by PI4K but not by PI3K activity. Activation of protein kinase A (PKA) by dibutyryl cAMP enhanced PIP(2(4,5)), PIP(3(3,4,5)), and F-actin formation, and these effects were mediated through PI3K. On the other hand, activation of PKC by phorbol myristate acetate enhanced PIP(2(4,5)) and F-actin formation mediated by PI4K activity, while the PI3K activity and intracellular PIP(3(3,4,5)) levels were reduced. These results suggest that two alternative pathways lead to PI4K activation: indirect activation by PKA, which is mediated by PI3K; and activation by PKC, which is independent of PI3K activity. Our results also suggest that spermine, which is present in the ejaculate, regulates PI4K activity during the capacitation process in vivo.     

A requirement of MAPKAPK2 in the uropod localization of PTEN during FMLP-induced neutrophil chemotaxis

The directionality control in chemotaxis is the result of a reciprocal regulation of PI3-kinase and PTEN subcellular localization. MK2(-/-) neutrophils have a directionality loss in fMLP-induced chemotaxis. We found that in polarized WT neutrophils PTEN was localized in the uropod region. However, MK2(-/-) neutrophils or p38 MAPK inhibitor-SB203580-pretreated WT neutrophils showed a disrupted PTEN subcellular localization. Some PTEN was localized at the leading edge of the polarized neutrophils, which may lower the concentration of PI3-kinase lipid product PtdIns(3,4,5)P3 required for directionality sensing. FMLP-stimulated MK2(-/-) neutrophils or SB203580-pretreated WT neutrophils also had disrupted F-actin polarization. F-actin polymerization inhibitor lantrunculin-B disrupted the polarization of PTEN, but not PtdIns(3,4,5)P3. The results suggest that PTEN uropod polarization is F-actin polymerization-dependent and may be through the effect of MK2 on F-actin polarization.     

A PLCβ/PI3Kγ-GSK3 signaling pathway regulates cofilin phosphatase slingshot2 and neutrophil polarization and chemotaxis

Neutrophils, in response to a chemoattractant gradient, undergo dynamic F-actin remodeling, a process important for their directional migration or chemotaxis. However, signaling mechanisms for chemoattractants to regulate the process are incompletely understood. Here, we characterized chemoattractant-activated signaling mechanisms that regulate cofilin dephosphorylation and actin cytoskeleton reorganization and are critical for neutrophil polarization and chemotaxis. In neutrophils, chemoattractants induced phosphorylation and inhibition of GSK3 via both PLCβ-PKC and PI3Kγ-AKT pathways, leading to the attenuation of GSK3-mediated phosphorylation and inhibition of the cofilin phosphatase slingshot2 and an increase in dephosphorylated, active cofilin. The relative contribution of this GSK3-mediated pathway to neutrophil chemotaxis regulation depended on neutrophil polarity preset by integrin-induced polarization of PIP5K1C. Therefore, our study characterizes a signaling mechanism for chemoattractant-induced actin cytoskeleton remodeling and elucidates its context-dependent role in regulating neutrophil polarization and chemotaxis.     

Regulation of SCFSKP2 Ubiquitin E3 Ligase Assembly and p27KIP1 Proteolysis by the PTEN Pathway and Cyclin D1

The PTEN tumor suppressor functions as a phosphatase of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and negatively regulates the PI 3-kinase signaling pathway. Our previous studies showed that PTEN expression causes accumulation of cyclin-dependent kinase inhibitor p27Kip1 and G1 cell cycle arrest. Here, we show that PTEN negatively regulates expression of cyclin D1 and that cyclin D1 plays a unique role in p27 proteolysis. Co-expression of cyclin D1, but not cyclin E, is sufficient to restore p27 levels in PTEN-expressing cells. Conversely, loss of cyclin D1 by siRNA causes p27 accumulation. Silencing of the cyclin D1 gene or inhibition of the PI 3-kinase pathway prevents formation of the SCFSKP2 complex, with a simultaneous increase in CUL1 binding to CAND1. CAND1-CUL1 binding is known to block the accessibility of CUL1 to SKP1 and SKP2. We have found that CUL1 is less neddylated in cells that have lost cyclin D1 expression. Using an in vitro extract system, we found that the extracts prepared from cells lacking cyclin D1 have reduced activity to neddylate CUL1, in a manner similar to extracts from cells treated with a PI 3-kinase inhibitor or in G0 resting cells. Consistenly, the steady state levels of CUL1 neddylation were found lower under these conditions. Our studies reveal that PTEN/PI 3-kinase signaling and cyclin D1 control a novel pathway that regulates assembly of the SCFSKP2 complex by modulating cullin neddylation and CAND1 binding at the G1/S cell cycle transition.