Protein Phosphatase 2A Isoform Research Articles

Protein Phosphatase 2A and Its Methylation Modulating Enzymes LCMT-1 and PME-1 Are Dysregulated in Tauopathies of Progressive Supranuclear Palsy and Alzheimer Disease.

Hyperphosphorylated tau aggregates are characteristic of tauopathies including progressive supranuclear palsy (PSP) and Alzheimer disease (AD), but factors contributing to pathologic tau phosphorylation are not well understood. Here, we studied the regulation of the major tau phosphatase, the heterotrimeric AB55αC protein phosphatase 2 A (PP2A), in PSP and AD. The assembly and activity of this PP2A isoform are regulated by reversible carboxyl methylation of its catalytic C subunit, while the B subunit confers substrate specificity. We sought to address whether the decreases in PP2A methylation and its methylating enzyme, leucine carboxyl methyltransferase (LCMT-1), which are reported in AD, relate to tau pathology or to concomitant amyloid pathology by comparing them in the relatively pure tauopathy PSP. Immunohistochemical analysis of frontal cortices showed that methyl-PP2A is reduced while demethyl-PP2A is increased, with no changes in total PP2A or B55α subunit, resulting in a reduction in the methyl/demethyl PP2A ratio of 63% in PSP and 75% in AD compared to controls. Similarly, Western blot analyses showed a decrease of methyl-PP2A and an increase of demethyl-PP2A with a concomitant reduction in the methyl/demethyl PP2A ratio in both PSP (74%) and AD (76%) brains. This was associated with a decrease in LCMT-1 and an increase in the demethylating enzyme, protein phosphatase methylesterase (PME-1), in both diseases. These findings suggest that PP2A dysregulation in tauopathies may contribute to the accumulation of hyperphosphorylated tau and to neurodegeneration.

Tumor Suppressor Role for B55 alpha PP2A Isoform in AML

Tumor Suppressor Role for B55 alpha PP2A Isoform in AML

The PP2A Subunit B55α Suppresses Survival Signaling in Acute Myeloid Leukemia Cells

Abstract 396Acute myeloid leukemia (AML) is still highly fatal so a better understanding of the mechanisms controlling chemoresistance is critical for the development of more effective therapies. The Protein Kinase B (AKT) regulates diverse cellular functions including protein translation, transcription, cell proliferation and apoptosis and is emerging as an important target for AML therapy. We have reported that activation of AKT predicts poor clinical outcome for patients with AML. Mutations of upstream activators of AKT such as Phosphatidylinositol 3 Kinase (PI3K) and RAS are well characterized and have been implicated in many cancers including AML. However, signal transduction is a dynamic process and the possibility arises that loss of protein phosphatase function can promote kinase activation in the absence of constitutive activation of the kinase by upstream activators. AKT has been shown to be regulated by Protein Phosphatase 2A (PP2A). PP2A is a family of hetero-trimeric serine/threonine phosphatases. While family members share a fairly common catalytic core (i.e. A and C subunits), the functional component of each PP2A isoform lies in its regulatory subunit (i.e. B subunit). Recent data suggests that suppression of PP2A can promote activation of survival signaling including the activation of AKT in AML. We recently identified that low expression of the PP2A isoform that acts as the AKT phosphatase (i.e. the one containing the B55α subunit) in AML negatively correlates with phosphorylation of AKT at threonine 308 and with AKT activation in a cohort of 511 AML patients (Ruvolo et al Leukemia, 2011). Patients with low B55α expression had shorter complete remission duration. In the current study, we now found by Reverse Phase Protein Analysis (RPPA) that there is a negative correlation between B55α expression and expression and phosphorylation of a number of survival kinases including Protein Kinase C a (PKCα) and SRC in AML patients. PP2A has been shown to negatively regulate the PKC family of kinases but this is the first report to implicate B55α in that process. While levels of phosphorylated PKCα negatively correlated with B55α, total levels of the kinase also were negatively correlated with the B subunit suggesting that phosphatase likely does not dephosphorylates PKCα but rather suppresses expression of the kinase. While SRC has not been linked to B55α, the kinase has been shown to negatively regulate the PP2A catalytic subunit (PP2A/C). Since PP2A is an obligate hetero-trimer, inactivation of PP2A/C via SRC could result in proteolytic cleavage of the B subunit. Consistent with this notion, treatment of OCI-AML3 cells with the tyrosine kinase inhibitor Dasatinib resulted in reduction of PP2A/C levels. Consistent with a mechanism whereby SRC regulated B55α rather than vice-versa, OCI-AML3 cells expressing B55α shRNA did not display changes in SRC phosphorylation status though there was a >67% loss of the B subunit in these cells. To determine the role of the AKT Phosphatase in AML cell survival, the OCI-AML3 cells expressing B55α shRNA were used to determine if reduction of levels of the phosphatase would promote AKT activation and promote chemoresistance. Cells with B55α shRNA exhibited higher levels of AKT S473 phosphorylation suggesting activation of the kinase. Furthermore, OCI-AML3 cells expressing B55α shRNA were more resistant to killing by the conventional chemotherapy agent AraC. One surprising finding was that OCI-AML3 B55α shRNA transductant cells were more resistant to okadaic acid, a PP2A inhibitor that is highly toxic to AML cells. Despite effects on AKT activation and survival, the reduction of the B subunit had no effect on total PP2A activity as determined by molybdate dye assay. Reduction of B55α did promote expression of at least one PP2A B regulatory subunit (i.e. B56α levels increased nearly 2 fold). Interestingly, B56α is positively regulated by AKT suggesting a possible feedback mechanism may exist between AKT and the various PP2A isoforms. An intriguing possibility arises that while PP2A is generally considered a tumor suppressor, inhibition of the AKT Phosphatase may activate an okadaic acid resistant PP2A isoform with survival function. Taken together, these results suggest that B55α may suppress survival signaling by kinases such as AKT and loss of function of the B subunit may promote resistance to chemotherapy in AML. Disclosures:No relevant conflicts of interest to declare.

505 Role for PP2A Regulatory Subunit B55alpha as a Regulator of AKT and Other Signaling Pathways in AML

Abstract Abstract 1668 Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures: No relevant conflicts of interest to declare.

Role for PP2A Regulatory Subunit B55α as A Regulator of AKT and Other Signaling Pathways In AML.

AbstractAbstract 1668Activation of survival kinases such as Protein Kinase B (AKT), Protein Kinase C (PKC), and Extracellular Receptor Activated Kinase (ERK) predict poor clinical outcome for patients with acute myeloid leukemia (AML; Kornblau et al Blood 2006). A better understanding of how the activities of these kinases are regulated by phosphorylation and dephosphorylation will enable the development of targeted therapies directed against this axis. Protein Phosphatase 2A (PP2A) negatively regulates PKC, AKT, and ERK but its role in AML is not clear. In the current study we examined the role of PP2A in regulating AKT in AML. Activation of AKT involves phosphorylation of threonine 308 (T308) and serine 473 (S473). A recent study has indicated that phosphorylation of AKT at T308 but not S473 is a poor prognostic factor for AML patients and that PP2A activity negatively correlated with T308 phosphorylation (Gallay et al Leukemia 2009). PP2A is a family of different isoforms that form hetero-trimers consisting of a catalytic C subunit, a scaffold A subunit, and one of at least 21 different regulatory B subunits. The functionality of each PP2A isoform is determined by the regulatory B subunit. Thus to understand PP2A regulation of AKT in AML, it is essential to study the B subunit that regulates the AKT phosphatase. The PP2A isoform regulating AKT in the AML patients is currently unknown. Evidence suggests that the B55a subunit is responsible for dephosphorylation of AKT at T308. In the current study, we compared B55α gene expression in blast cells derived from AML patients with normal counterpart (i.e. CD34+) cells derived from normal bone marrow donors by real time PCR. Surprisingly, B55α gene expression was higher in the patients. Reverse Phase Protein Analysis (RPPA) is a powerful tool that allows for the analysis of protein expression from patient samples. Protein levels of the PP2A B subunit were analyzed by RPPA in AML blast cells obtained from 511 newly diagnosed AML patients and CD34+ cells obtained from 11 normal bone marrow donors. Levels of B55α protein were significantly lower in the blast cells from the AML patients compared to normal CD34+ cells. While the mechanism for the observed difference in gene versus protein expression in the leukemia cells has yet to be determined, a plausible mechanism is that the B55α protein is being proteolyzed since monomeric PP2A B subunits that are not part of the PP2A hetero-trimer are degraded. Importantly the reduced levels of B55α protein observed would be predicted if AKT were activated in the AML blast cells. We next compared AKT phosphorylation status with B55α protein expression in the AML blast cells using RPPA to answer this question. Analysis of RPPA data revealed that there was no correlation between B55α protein levels and levels of total AKT protein or with levels of AKT phosphorylated at S473 in the AML samples. However, there was a moderate but significant negative correlation between B55α protein levels and levels of AKT phosphorylated at T308. This result suggests that B55α is mediating dephosphorylation of AKT at T308 but not S473 in the AML cells. B55α expression was not associated with FAB classification but was positively correlated with high blast and peripheral blood counts. While the level of expression of the B subunit did not correlate with overall survival, intermediate levels of B55α expression were associated with longer complete remission duration. We predict that higher levels of B55α would reflect low levels of other PP2A B subunits. Consistent with this prediction, B55α expression positively correlated with MYC expression in the AML patients. MYC expression is regulated by a B subunit that competes with B55α (i.e. B56α). These findings suggest that B55α may play an important role in AML as a negative regulator of AKT and perhaps by other as yet unidentified functions. Activation of B55α is a potential therapeutic target for overcoming the AKT activation frequently observed in AML. Disclosures:No relevant conflicts of interest to declare.

PKR Promotes BCL2 Phosphatase Function in ALL Cells by Dual Mechanisms: As a B56α Kinase and by Supporting B56α Expression Via eIF2α And the PI3K/AKT Cascade.

Activation of double-stranded RNA-activated Protein Kinase (PKR) is associated with growth inhibition and cell death. PKR is normally dormant in cells and is activated in response to stress challenge (e.g. viral infection, chemotherapy, factor withdrawal). The presence of active kinase under growth conditions is generally not observed. PKR expression and activation status was determined in four ALL cell lines: REH, CCRF-CEM, MOLT4, and RS(4;11). PKR was found to be expressed and phosphorylated in all the cell lines. The presence of active PKR suggests the kinase may be important for cellular homeostasis in ALL cells. The best characterized substrate of PKR is eIF2α. Phosphorylation of eIF2α was examined to determine if PKR phosphorylation levels correlated with phosphorylation of the kinase's primary substrate. Phosphorylated eIF2α was detected in all the cell lines except RS(4;11) cells. The lack of observed phosphorylated eIF2α in the RS(4;11) cells was due to the low abundance of the eIF2α protein. Another substrate of PKR is B56α, a B regulatory subunit of Protein Phosphatase 2A (PP2A). B56α comprises a mitochondrial PP2A isoform that serves as the BCL2 phosphatase and promotes cell death in response to chemotherapeutic agents. PKR was found to phosphorylate B56α at serine 28 in REH cells to promote mitochondrial PP2A activity and BCL2 phosphatase function. Loss of PKR by shRNA results in phosphorylation of BCL2 and the cells become resistant to the chemotherapeutic drug etoposide. Inhibition of PKR by pharmacologic inhibitor or shRNA in REH cells results in loss of B56α expression suggesting that phosphorylation may be required for stability of the B subunit. However, phosphorylation of B56α is not required for its stability since mutant S28A B56α protein is readily expressed in cells. A clue to an alternative mechanism was provided by the eIF2α phosphorylation status and B56α expression pattern in the four ALL cell lines. RS(4;11) cells display little if any B56α protein while the other three ALL cell lines express the B subunit. REH, CCRF-CEM, and MOLT4 cells exhibited ~ 3X higher mitochondrial PP2A activity compared to RS(4;11) cells. The lack of mitochondrial PP2A activity in RS(4;11) cells is likely due to a lack of B56α. Since RS(4;11) cells have active PKR but lack phosphorylated eIF2α, the possibility arises that B56α expression may depend on phosphorylated eIF2α. The mechanism how PKR promotes B56α expression appears to involve the proteasome since a proteasome inhibitor can block loss of the B subunit when PKR is suppressed. A role for eIF2α is suggested since salubrinal, a drug that promotes eIF2α phosphorylation by preventing the dephosphorylation of the molecule increased expression of B56α protein. It has recently been found that eIF2α phosphorylation can activate the PI3K/AKT signaling cascade. Suppression of PKR in REH cells blocks AKT phosphorylation suggesting that PKR may positively regulate AKT in these cells. It appears that the mechanism how PKR promotes B56α expression involves the PI3K/AKT cascade since inhibition of this signaling pathway using LY294002 blocks expression of the B subunit. Since B56α mediated mitochondrial PP2A activity promotes cell death in response to chemotherapeutic agents, it is likely that the normally pro-survival PI3K/AKT pathway may serve a role in stress signaling in ALL cells by supporting B56α expression to promote BCL2 phosphatase activity. These findings indicate that PKR can regulate the BCL2 phosphatase at multiple levels in ALL cells. Understanding the complex regulatory pathway how PKR controls PP2A and BCL2 will be necessary for designing effective therapies for the treatment of ALL.

The B56α PP2A B Regulatory Subunit Promotes Mitochondrial PP2A Activity and Supports Etoposide-Induced Cell Death in Human ALL Derived REH Cells.

Protein Phosphatase 2A (PP2A) has broad regulatory effects on diverse signaling pathways so it is not surprising that PP2A is emerging as a possible tumor suppressor. The PP2A inhibitor okadaic acid has been found to promote tumors. Cellular transformation caused by viruses has been shown to involve dysregulation of PP2A pathways (e.g. the SV40 small T antigen). Mutations in PP2A subunit genes have been reported in lung cancer and breast cancer. PP2A does have a role in chronic myeloid leukemia (CML) tumorigenesis. While a role for PP2A in CML is emerging, the significance of PP2A signaling pathways in acute lymphoblastic leukemia (ALL) is currently not clear. PP2A has been shown to negatively regulate BCL2 function in human ALL derived REH cells. Considering that BCL2 is a potent anti-apoptotic molecule, a possibility arises that PP2A may regulate chemoresistance in ALL cells via a mechanism involving BCL2. Supporting such a notion, low dose okadaic acid treatment of REH cells results in robust BCL2 phosphorylation and promotes resistance to chemotherapeutic drugs such as etoposide. A difficulty in studying PP2A-mediated signaling is that the enzyme has a multimeric structure. PP2A is a heterotrimer comprising a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). There are 2 highly homologous isoforms of the A subunit, 2 highly homologous isoforms of the C subunit, and there are 3 major B subunit families (i.e. B55, B56, and PR72) that at present include 21 proteins. Recent studies indicate that PP2A substrate specificity and sub-cellular localization are mediated by the B subunit. Thus PP2A is not a single enzyme but rather a family of protein phosphatase isoforms defined by which B regulatory subunit controls its function. Little is known about the mechanisms regulating B subunit function. It has been found that a ceramide activated mitochondrial PP2A isoform containing the B56 family member B56α acts as the BCL2 phosphatase in REH cells. In the present study, it was found that over-expression of exogenous B56α in REH cells promoted sensitivity to the chemotherapeutic drug etoposide. B56α was found to promote mitochondrial but not nuclear PP2A activity. B56α was found to promote dephosphorylation of PKCα but not PKCε. This finding suggests that regulation of PKC signaling by PP2A is dependent on different B subunits. As PKCα is a physiologic BCL2 kinase, the ability for B56α to control the PP2A isoform that targets both BCL2 and at least one BCL2 kinase suggests PP2A regulation of BCL2 function is multi-tiered. Finally, over-expression of B56α promoted changes in the composition of proteins present in the mitochondrial membranes as determined by 2D gel electrophoresis. A broad range of proteins were shown to be affected by B56α over-expression (mitochondrial proteins varied in size from < 18kd to > 100kd), PP2A stress signaling mediated by B56α may involve a number of targets. These findings suggest that B56α regulation of PP2A stress signaling is complex but an understanding of this process may result in new strategies for the treatment of ALL.

Molecular basis for PP2A regulatory subunit B56α targeting in cardiomyocytes

Protein phosphatase 2A (PP2A) is a multifunctional protein phosphatase with critical roles in excitable cell signaling. In the heart, PP2A function is linked with modulation of beta-adrenergic signaling and has been suggested to regulate key ion channels and transporters including Na/Ca exchanger, ryanodine receptor, inositol 1,4,5-trisphosphate receptor, and Na/K ATPase. Although many of the functional roles and molecular targets for PP2A in heart are known, little is established regarding the cellular pathways that localize specific PP2A isoform activities to subcellular sites. We report that the PP2A regulatory subunit B56alpha is an in vivo binding partner for ankyrin-B, an adapter protein required for normal subcellular localization of the Na/Ca exchanger, Na/K ATPase, and inositol 1,4,5-trisphosphate receptor. Ankyrin-B and B56alpha are colocalized and coimmunoprecipitate in primary cardiomyocytes. Using multiple strategies, we identified the structural requirements on B56alpha for ankyrin-B association as a 13 residue motif in the B56alpha COOH terminus not present in other B56 family polypeptides. Finally, we report that reduced ankyrin-B expression in primary ankyrin-B(+/-) cardiomyocytes results in disorganized distribution of B56alpha that can be rescued by exogenous expression of ankyrin-B. These new data implicate ankyrin-B as a critical targeting component for PP2A in heart and identify a new class of signaling proteins targeted by ankyrin polypeptides.

Regulation of hepatic protein phosphatase 2A (PP2A): Role of catalytic subunit methylation

PP2A catalytic subunit (C) associates with multiple regulatory proteins. One, alpha4 (a4), is a downstream effector of mTOR. We studied PP2A regulation in two models of liver growth, rapamycin-sensitive adult hepatocyte proliferation after partial hepatectomy (PH) and rapamycin-resistant late gestation fetal hepatocyte proliferation. Immunoblotting of fetal and adult liver homogenates with antibody toward full-length C showed higher levels in fetal samples. In contrast, carboxy-terminus antibodies showed a more intense signal in adult samples. This discrepancy was explained by high levels of methylated C (Me-C) in fetal liver. MonoQ fractionation of homogenates showed that demethylated C distributed to A-subunit/C (AC) dimers. Me-C predominated in ABC heterotrimers and in a4/C dimers. Adult liver showed slightly higher levels of a4/C, possibly due to lower levels of competing A-subunit. Neither PH nor rapamycin administration altered content of AC, ABC, a4/C or Me-C. Our results indicate that a4/C abundance is not directly regulated by mTOR in liver. However, PP2A-C methylation is developmentally regulated and determines PP2A isoform distribution, thus indicating its critical regulatory role.

Multiple effects of protein phosphatase 2A on nutrient-induced signalling in the yeast Saccharomyces cerevisiae.

The trehalose-degrading enzyme trehalase is activated upon addition of glucose to derepressed cells or in response to nitrogen source addition to nitrogen-starved glucose-repressed yeast (Saccharomyces cerevisiae) cells. Trehalase activation is mediated by phosphorylation. Inactivation involves dephosphorylation, as trehalase protein levels do not change upon multiple activation/inactivation cycles. Purified trehalase can be inactivated by incubation with protein phosphatase 2A (PP2A) in vitro. To test whether PP2A was involved in trehalase inactivation in vivo, we overexpressed the yeast PP2A isoform Pph22. Unexpectedly, the moderate (approximately threefold) overexpression of Pph22 that we obtained increased basal trehalase activity and rendered this activity unresponsive to the addition of glucose or a nitrogen source. Concomitant with higher basal trehalase activity, cells overexpressing Pph22 did not store trehalose efficiently and were heat sensitive. After the addition of glucose or of a nitrogen source to starved cells, Pph22-overexpressing cells showed a delayed exit from stationary phase, a delayed induction of ribosomal gene expression and constitutive repression of stress-regulated element-controlled genes. Deletion of the SCH9 gene encoding a protein kinase involved in nutrient-induced signal transduction restored glucose-induced trehalase activation in Pph22-overexpressing cells. Taken together, our results indicate that yeast PP2A overexpression leads to the activation of nutrient-induced signal transduction pathways in the absence of nutrients.

Regulation of endosome sorting by a specific PP2A isoform.

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869-5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205-216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.