Sphingomyelin synthases regulate production of diacylglycerol at the Golgi

SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.     

Adipsin and the glucose transporter GLUT4 traffic to the cell surface via independent pathways in adipocytes

Insulin increases the exocytosis of many soluble and membrane proteins in adipocytes. This may reflect a general effect of insulin on protein export from the trans Golgi network. To test this hypothesis, we have compared the trafficking of the secreted serine protease adipsin and the integral membrane proteins GLUT4 and transferrin receptors in 3T3-L1 adipocytes. We show that adipsin is secreted from the trans Golgi network to the endosomal system, as ablation of endosomes using transferrin-HRP conjugates strongly inhibited adipsin secretion. Phospholipase D has been implicated in export from the trans Golgi network, and we show that insulin stimulates phospholipase D activity in these cells. Inhibition of phospholipase D action with butan-1-ol blocked adipsin secretion and resulted in accumulation of adipsin in trans Golgi network-derived vesicles. In contrast, butan-1-ol did not affect the insulin-stimulated movement of transferrin receptors to the plasma membrane, whereas this was abrogated following endosome ablation. GLUT4 trafficking to the cell surface does not utilise this pathway, as insulin-stimulated GLUT4 translocation is still observed after endosome ablation or inhibition of phospholipase D activity. Immunolabelling revealed that adipsin and GLUT4 are predominantly localised to distinct intracellular compartments. These data suggest that insulin stimulates the activity of the constitutive secretory pathway in adipocytes possibly by increasing the budding step at the TGN by a phospholipase D-dependent mechanism. This may have relevance for the secretion of other soluble molecules from these cells. This is not the pathway employed to deliver GLUT4 to the plasma membrane, arguing that insulin stimulates multiple pathways to the cell surface in adipocytes.     

PKCeta is required for beta1gamma2/beta3gamma2- and PKD-mediated transport to the cell surface and the organization of the Golgi apparatus

Protein kinase D (PKD) binds to a pool of diacylglycerol (DAG) in the TGN and undergoes a process of activation that involves heterotrimeric GTP-binding protein subunits betagamma to regulate membrane fission. This fission reaction is used to generate transport carriers at the TGN that are en route to the cell surface. We now report that PKD is activated specifically by G protein subunit beta1gamma2 and beta3gamma2 via the Golgi apparatus-associated PKCeta. Compromising the kinase activity of PKCeta-inhibited protein transport from TGN to the cell surface. Expression of constitutively activated PKCeta caused Golgi fragmentation, which was inhibited by a kinase inactive form of PKD. Our findings reveal that betagamma, PKCeta, and PKD act in series to generate transport carriers from the TGN and their overactivation results in complete vesiculation of the Golgi apparatus.     

Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network

Protein kinase D (PKD) binds to diacylglycerol (DAG) in the trans-Golgi network (TGN) and is activated by trimeric G-protein subunits beta gamma. This complex then regulates the formation of transport carriers in the TGN that traffic to the plasma membrane in non-polarized cells. Here we report specificity of different PKD isoforms in regulating protein trafficking from the TGN. Kinase-inactive forms of PKD1, PKD2 and PKD3 localize to the TGN in polarized and non-polarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.     

Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gbetagamma (GTP-binding protein betagamma subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCeta (protein kinase Ceta) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCeta, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gbetagamma, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that betagamma-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCbeta3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCbeta3, which is necessary to activate PKCeta and PKD in that Golgi compartment, via DAG production.     

Dimeric PKD regulates membrane fission to form transport carriers at the TGN

Protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) through interaction with diacylglycerol (DAG) and is required for the biogenesis of TGN to cell surface transport carriers. We now provide definitive evidence that PKD has a function in membrane fission. PKD depletion by siRNA inhibits trafficking from the TGN, whereas expression of a constitutively active PKD converts TGN into small vesicles. These findings demonstrate that PKD regulates membrane fission and this activity is used to control the size of transport carriers, and to prevent uncontrolled vesiculation of TGN during protein transport.     

Protein kinase D2 regulates chromogranin A secretion in human BON neuroendocrine tumour cells

Chromogranin A is a member of the granin family of acidic secretory glycoproteins that is found in secretory granules of many endocrine cells including neuroendocrine tumour cells. This hormone serves as a model system for autonomous hormone secretion by the so called functional neuroendocrine tumours of the gastrointestinal tract. The precise regulation of chromogranin secretion at the level of the Golgi apparatus is a subject of intense research. The protein kinase D (PKD) family of serine threonine kinases has so far been implicated in the regulation of constitutive secretion in epithelial cells. Here we examined whether PKD2 expression and activity could also play a role in the release of secretory granules from the trans Golgi network (TGN) in neuroendocrine tumour cells and hence be a target to block autonomous secretion by these tumours. Our data show that expression and catalytic activity of PKD2 are required for the release of chromogranin A containing secretory vesicles. Inhibition of PKD2 activity or siRNA knockdown of PKD2 resulted in a marked perinuclear retention of chromogranin A immunofluorescence in the trans Golgi network and led to a marked reduction in basal as well as phorbol ester stimulated secretion of chromogranin A into the supernatant of cells. Thus, PKD2 controls the release of secretory granules in neuroendocrine tumour cells at the level of the Golgi apparatus and could hence serve as a novel target to block hormone secretion in functional neuroendocrine tumours.     

Recruitment of arfaptins to the trans‐Golgi network by PI(4)P and their involvement in cargo export

The BAR (Bin/Amphiphysin/Rvs) domain proteins arfaptin1 and arfaptin2 are localized to the trans‐Golgi network (TGN) and, by virtue of their ability to sense and/or generate membrane curvature, could play an important role in the biogenesis of transport carriers. We report that arfaptins contain an amphipathic helix (AH) preceding the BAR domain, which is essential for their binding to phosphatidylinositol 4‐phosphate (PI(4)P)‐containing liposomes and the TGN of mammalian cells. The binding of arfaptin1, but not arfaptin2, to PI(4)P is regulated by protein kinase D (PKD) mediated phosphorylation at Ser100 within the AH. We also found that only arfaptin1 is required for the PKD‐dependent trafficking of chromogranin A by the regulated secretory pathway. Altogether, these findings reveal the importance of PI(4)P and PKD in the recruitment of arfaptins at the TGN and their requirement in the events leading to the biogenesis of secretory storage granules.     

Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion

Vesicle formation and fission are tightly regulated at the trans-Golgi network (TGN) during constitutive secretion. Two major protein families regulate these processes: members of the adenosyl-ribosylation factor family of small G-proteins (ARFs) and the protein kinase D (PKD) family of serine/threonine kinases. The functional relationship between these two key regulators of protein transport from the TGN so far is elusive. We here demonstrate the assembly of a novel functional protein complex at the TGN and its key members: cytosolic PKD2 binds ARF-like GTPase (ARL1) and shuttles ARL1 to the TGN. ARL1, in turn, localizes Arfaptin2 to the TGN. At the TGN, where PKD2 interacts with active ARF1, PKD2, and ARL1 are required for the assembly of a complex comprising of ARF1 and Arfaptin2 leading to secretion of matrix metalloproteinase-2 and -7. In conclusion, our data indicate that PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo.     

Roles for the Drs2p-Cdc50p complex in protein transport and phosphatidylserine asymmetry of the yeast plasma membrane

Drs2p, a P-type adenosine triphosphatase required for a phosphatidylserine (PS) flippase activity in the yeast trans Golgi network (TGN), was first implicated in protein trafficking by a screen for mutations synthetically lethal with arf1 (swa). Here, we show that SWA4 is allelic to CDC50, encoding a membrane protein previously shown to chaperone Drs2p from the endoplasmic reticulum to the Golgi complex. We find that cdc50Delta exhibits the same clathrin-deficient phenotypes as drs2Delta, including delayed transport of carboxypeptidase Y to the vacuole, mislocalization of resident TGN enzymes and the accumulation of aberrant membrane structures. These trafficking defects precede appearance of cell polarity defects in cdc50Delta, suggesting that the latter are a secondary consequence of disrupting Golgi function. Involvement of Drs2p-Cdc50p in PS translocation suggests a role in restricting PS to the cytosolic leaflet of the Golgi and plasma membrane. Annexin V binding and papuamide B hypersensitivity indicate that drs2Delta or cdc50Delta causes a loss of plasma membrane PS asymmetry. However, clathrin and other endocytosis null mutants also exhibit a comparable loss of PS asymmetry, and studies with drs2-ts and clathrin (chc1-ts) conditional mutants suggest that loss of plasma membrane asymmetry is a secondary consequence of disrupting protein trafficking.     

Protein kinase D 3 is localized in vesicular structures and interacts with vesicle-associated membrane protein 2

Protein kinase D localizes in the Golgi and regulates protein transport from the Golgi to the plasma membrane. In the present study, we found that PKD3, a novel member of the PKD family, and its fluorescent protein fusions localized in the Golgi and in the vesicular structures that are in part marked by endosome markers. Fluorescent recovery after photobleaching (FRAP) showed that the PKD3-associated vesicular structures were constantly forming and dissolving, reflecting active subcellular structures. FRAP on plasma membrane-located PKD3 indicated a slower recovery of PKD3 fluorescent signal compared to those of PKC isoforms, implying a different targeting mechanism at the plasma membrane. VAMP2, the vesicle-localized v-SNARE, was later identified as a novel binding partner of PKD3 through yeast two-hybrid screening. PKD3 directly interacted with VAMP2 in vitro and in vivo, and colocalized in part with VAMP2 vesicles in cells. PKD3 did not phosphorylate VAMP-GFP and the purified GST-VAMP2 protein in in vitro phosphorylation assays. Rather, PKD3 was found to promote the recruitment of VAMP2 vesicles to the plasma membrane in response to PMA, while the kinase dead PKD3 abolished this effect. Thus, the kinase activity of PKD3 was required for PMA-induced plasma membrane trafficking of VAMP2. In summary, our findings suggest that PKD3 localizes to vesicular structures that are part of the endocytic compartment. The vesicular distribution may be attributed in part to the direct interaction between PKD3 and vesicle-associated membrane protein VAMP2, through which PKD3 may regulate VAMP2 vesicle trafficking by facilitating its recruitment to the target membrane.     

PKD Regulates Membrane Fission to Generate TGN to Cell Surface Transport Carriers

The serine/threonine protein kinase D (PKD) is recruited to the trans-Golgi network (TGN) by binding diacylglycerol (DAG) and the ARF1 GTPase. PKD, at the TGN, promotes the production of phosphatidylinositol-4 phosphate (PI4P) by activating the lipid kinase phophatidylinositol 4-kinase IIIß (PI4KIIIß). PI4P recruits proteins such as oxysterol-binding protein 1 (OSBP) and ceramide transport protein (CERT) that control sphingolipid and sterol levels at the TGN. CERT mediated transport of ceramide to the TGN, we suggest, is used for increasing the local production and concentration of DAG. Once the crucial concentration of DAG is achieved, OSBP and CERT dissociate from the TGN on phosphorylation by PKD and DAG is sequentially converted into phosphatidic acid (PA) and lyso-PA (LPA). Therefore, the net effect of the activated PKD at the TGN is the sequential production of the modified lipids DAG, PA, and LPA that are necessary for membrane fission to generate cell surface specific transport carriers.     

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.     

A Golgi PKD Activity Reporter Reveals a Crucial Role of PKD in Nocodazole-Induced Golgi Dispersal

The protein kinase D (PKD) family comprises multifunctional serine/threonine-specific protein kinases with three mammalian isoforms: PKD1, PKD2 and PKD3. A prominent PKD function is the regulation of basolateral-targeted transport carrier fission from the trans -Golgi network (TGN). To visualize site-specific PKD activation at this organelle, we designed a molecular reporter consisting of a PKD-specific substrate sequence fused to enhanced green fluorescent protein (EGFP), specifically targeted to the TGN via the p230 GRIP domain. Quantitative analyses using a phosphospecific antibody and ratiometric fluorescence imaging revealed that Golgi-specific phosphorylation of the reporter was strictly dependent on stimulation of endogenous PKD or transient expression of active PKD constructs. Conversely, PKD-specific pharmacological inhibitors and siRNA-mediated PKD knockdown suppressed reporter phosphorylation. Using this reporter we investigated a potential role for PKD in the regulation of Golgi complex morphology. Interestingly, nocodazole-induced Golgi complex break-up and dispersal was associated with local PKD activation as measured by reporter phosphorylation and this was efficiently blocked by expression of a dominant-negative PKD mutant or PKD depletion. Our data thus identify a novel link between PKD activity and the microtubule cytoskeleton, whereby Golgi complex integrity is regulated.     

Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility

BACKGROUND: Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.RESULTS: To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.CONCLUSIONS: These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.     

Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 (kAE1) to the Cell Surface

Kidney anion exchanger 1 (kAE1) plays an important role in acid–base homeostasis by mediating chloride/bicarbornate (Cl^?/HCO₃^?) exchange at the basolateral membrane of α‐intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease – distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans‐Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral‐related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non‐polarized kidney cells. By using RNA interference, co‐immunoprecipitation, yellow fluorescent protein‐based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin (but not AP‐1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral‐related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP‐1 mu1A, AP‐3 mu1, AP‐4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid‐secreting α‐intercalated cells.      

Cog2 null mutant CHO cells show defective sphingomyelin synthesis

The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ～25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.     

Phosphoinositide 3-kinase �� regulates membrane fission of Golgi carriers for selective cytokine secretion

Phosphoinositide 3-kinase (PI3K) p110 isoforms are membrane lipid kinases classically involved in signal transduction. Lipopolysaccharide (LPS)-activated macrophages constitutively and abundantly secrete proinflammatory cytokines including tumor necrosis factor-α (TNF). Loss of function of the p110δ isoform of PI3K using inhibitors, RNA-mediated knockdown, or genetic inactivation in mice abolishes TNF trafficking and secretion, trapping TNF in tubular carriers at the trans-Golgi network (TGN). Kinase-active p110δ localizes to the Golgi complex in LPS-activated macrophages, and TNF is loaded into p230-labeled tubules, which cannot undergo fission when p110δ is inactivated. Similar blocks in fission of these tubules and in TNF secretion result from inhibition of the guanosine triphosphatase dynamin 2. These findings demonstrate a new function for p110δ as part of the membrane fission machinery required at the TGN for the selective trafficking and secretion of cytokines in macrophages.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.