Mitotic Phosphoproteins Research Articles

A novel HDAC6 inhibitor interferes microtubule dynamics and spindle assembly checkpoint and sensitizes cisplatin-induced apoptosis in castration-resistant prostate cancer.

Metastatic castration-resistant prostate cancer (CRPC), the most refractory prostate cancer, inevitably progresses and becomes unresponsive to hormone therapy, revealing a pressing unmet need for this disease. Novel agents targeting HDAC6 and microtubule dynamics can be a potential anti-CRPC strategy. Cell proliferation was examined in CRPC PC-3 and DU-145 cells using sulforhodamine B assay and anchorage-dependent colony formation assay. Flow cytometric analysis of propidium iodide staining was used to determine cell-cycle progression. Cell-based tubulin polymerization assay and confocal immunofluorescence microscopic examination determine microtubule assembly/disassembly status. Protein expressions were determined using Western blot analysis. A total of 82 novel derivatives targeting HDAC6 were designed and synthesized, and Compound 25202 stood out, showing the highest efficacy in blocking HDAC6 (IC50, 3.5 nM in enzyme assay; IC50, 1.0 μM in antiproliferative assay in CRPC cells), superior to tubastatin A (IC50, 5.4 μM in antiproliferative assay). The selectivity and superiority of 25202 were validated by examining the acetylation of both α-tubulin and histone H3, detecting cell apoptosis and HDACs enzyme activity assessment. Notably, 25202 but not tubastatin A significantly decreased HDAC6 protein expression. 25202 prolonged mitotic arrest through the detection of cyclin B1 upregulation, Cdk1 activation, mitotic phosphoprotein levels, and Bcl-2 phosphorylation. Compound 25202 did not mimic docetaxel in inducing tubulin polymerization but disrupted microtubule organization. Compound 25202 also increased the phosphorylation of CDC20, BUB1, and BUBR1, indicating the activation of the spindle assembly checkpoint (SAC). Moreover, 25202 profoundly sensitized cisplatin-induced cell death through impairment of cisplatin-evoked DNA damage response and DNA repair in both ATR-Chk1 and ATM-Chk2 pathways. The data suggest that 25202 is a novel selective and potent HDAC6 inhibitor. Compound 25202 blocks HDAC6 activity and interferes microtubule dynamics, leading to SAC activation and mitotic arrest prolongation that eventually cause apoptosis of CRPC cells. Furthermore, 25202 sensitizes cisplatin-induced cell apoptosis through impeding DNA damage repair pathways.

Protein Phosphatases Involved in Regulating Mitosis: Facts and Hypotheses.

Almost all eukaryotic proteins are subject to post-translational modifications during mitosis and cell cycle, and in particular, reversible phosphorylation being a key event. The recent use of high-throughput experimental analyses has revealed that more than 70% of all eukaryotic proteins are regulated by phosphorylation; however, the mechanism of dephosphorylation, counteracting phosphorylation, is relatively unknown. Recent discoveries have shown that many of the protein phosphatases are involved in the temporal and spatial control of mitotic events, such as mitotic entry, mitotic spindle assembly, chromosome architecture changes and cohesion, and mitotic exit. This implies that certain phosphatases are tightly regulated for timely dephosphorylation of key mitotic phosphoproteins and are essential for control of various mitotic processes. This review describes the physiological and pathological roles of mitotic phosphatases, as well as the versatile role of various protein phosphatases in several mitotic events.

KUD773, a phenylthiazole derivative, displays anticancer activity in human hormone-refractory prostate cancers through inhibition of tubulin polymerization and anti-Aurora A activity

BackgroundHormone-refractory prostate cancer (HRPC), which is resistant to hormone therapy, is a major obstacle in clinical treatment. An approach to inhibit HRPC growth and ultimately to kill cancers is highly demanded.ResultsKUD773 induced the anti-proliferative effect and subsequent apoptosis in PC-3 and DU-145 (two HRPC cell lines); whereas, it showed less active in normal prostate cells. Further examination showed that KUD773 inhibited tubulin polymerization and induced an increase of mitotic phosphoproteins and polo-like kinase 1 (PLK1) phosphorylation, indicating a mitotic arrest of the cell cycle through an anti-tubulin action. The kinase assay demonstrated that KUD773 inhibited Aurora A activity. KUD773 induced an increase of Cdk1 phosphorylation at Thr161 (a stimulatory phosphorylation site) and a decrease of phosphorylation at Tyr15 (an inhibitory phosphorylation site), suggesting the activation of Cdk1. The data were substantiated by an up-regulation of cyclin B1 (a Cdk1 partner). Furthermore, KUD773 induced the phosphorylation and subsequent down-regulation of Bcl-2 and activation of caspase cascades.ConclusionsThe data suggest that KUD773 induces apoptotic signaling in a sequential manner. It inhibits tubulin polymerization associated with an anti-Aurora A activity, leading to Cdk1 activation and mitotic arrest of the cell cycle that in turn induces Bcl-2 degradation and a subsequent caspase activation in HRPCs.

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

Bim vanishes in the light of a mitotic Aurora

Bim vanishes in the light of a mitotic Aurora

Aurora‐A‐elicited Astrin phosphorylation regulates mitotic progression

Astrin is a spindle protein regulating several aspects of mitosis including the maintenance of bipolar spindle pole, regulation of sisterchromatid cohesion and chromosome alignment. However, it remains unclear how to regulate Astrin protein in mitosis. Our present study has shown that Astrin behaves as a mitotic phosphoprotein and was directly phosphorylated by the mitotic kinase Aurora‐A in an in vitro kinase assay. Furthermore, the mitosis dependent phosphorylation of Astrin was blockaded by immunodepletion or shRNA‐mediated knockdown of Aurora‐A. It indicates that Astrin acts as an in vitro and in vivo substrate of Aurora‐A. The phosphorylation site was subsequently mapped to serine 115 of Astrin. And site‐directed mutagenesis analysis revealed that Astrin S115A mutants, no longer serving as a substrate of Aurora‐A, delayed the mitosis progression and induced chromosome misalignment and DNA lagging along the mitotic progression. By contrast, Astrin S115D, which mimics Aurora‐A elicited phosphorylation, accelerated the mitosis progression and slightly deceased the level of DNA lagging. The binding activity of Astrin S115A mutant to its mitotic interaction proteins LC8 and CLASP‐1alpha was remarkably reduced, leading to the mislocalization of the sequential two proteins and the misalignment of chromosome. In conclusion, we are the first to demonstrate that Aurora‐A, a well‐characterized oncokinase controlling a wide range of mitotic events by phosphorylating numerous mitotic factors, phosphorylates Astrin in vitro and in vivo, which guides the binding of Astrin to its cellular partners, and ensures accurate chromosome alignment and segregation and thereby proper mitosis progression.

BUBR1 recruits PP2A via the B56 family of targeting subunits to promote chromosome congression

SummaryBUBR1 is a mitotic phosphoprotein essential for the maintenance of chromosome stability by promoting chromosome congression and proper kinetochore–microtubule (K-fiber) attachment, but the underlying mechanism(s) has remained elusive. Here we identify BUBR1 as a binding partner of the B56 family of Protein Phosphatase 2A regulatory subunits. The interaction between BUBR1 and the B56 family is required for chromosome congression, since point mutations in BUBR1 that block B56 binding abolish chromosome congression. The BUBR1:B56-PP2A complex opposes Aurora B kinase activity, since loss of the complex can be reverted by inhibiting Aurora B. Importantly, we show that the failure of BUBR1 to recruit B56-PP2A also contributes to the chromosome congression defects found in cells derived from patients with the Mosaic Variegated Aneuploidy (MVA) syndrome. Together, we propose that B56-PP2A is a key mediator of BUBR1's role in chromosome congression and functions by antagonizing Aurora B activity at the kinetochore for establishing stable kinetochore–microtubule attachment at the metaphase plate.

Non-catalytic participation of the Pin1 peptidyl-prolyl isomerase domain in target binding

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase (PPIase) that has the potential to add an additional level of regulation within protein kinase mediated signaling pathways. Furthermore, there is a mounting body of evidence implicating Pin1 in the emergence of pathological phenotypes in neurodegeneration and cancer through the isomerization of a wide variety of substrates at peptidyl-prolyl bonds where the residue preceding proline is a phosphorylated serine or threonine residue (i.e., pS/T-P motifs). A key step in this regulatory process is the interaction of Pin-1 with its substrates. This is a complex process since Pin1 is composed of two domains, the catalytic PPIase domain, and a type IV WW domain, both of which recognize pS/T-P motifs. The observation that the WW domain exhibits considerably higher binding affinity for pS/T-P motifs has led to predictions that the two domains may have distinct roles in mediating the actions of Pin1 on its substrates. To evaluate the participation of its individual domains in target binding, we performed GST pulldowns to monitor interactions between various forms of Pin1 and mitotic phospho-proteins that revealed two classes of Pin-1 interacting proteins, differing in their requirement for residues within the PPIase domain. From these observations, we consider models for Pin1-substrate interactions and the potential functions of the different classes of Pin1 interacting proteins. We also compare sequences that are recognized by Pin1 within its individual interaction partners to investigate the underlying basis for its different types of interactions.

A Framework for Image-Based Classification of Mitotic Cells in Asynchronous Populations

High content screening (HCS) has emerged an important tool for drug discovery because it combines rich readouts of cellular responses in a single experiment. Inclusion of cell cycle analysis into HCS is essential to identify clinically suitable anticancer drugs that disrupt the aberrant mitotic activity of cells. One challenge for integration of cell cycle analysis into HCS is that cells must be chemically synchronized to specific phases, adding experimental complexity to high content screens. To address this issue, we have developed a rules-based method that utilizes mitotic phosphoprotein monoclonal 2 (MPM-2) marker and works consistently in different experimental conditions and in asynchronous populations. Further, the performance of the rules-based method is comparable to established machine learning approaches for classifying cell cycle data, indicating the robustness of the features we use in the framework. As such, we suggest the use of MPM-2 analysis and its associated expressive features for integration into HCS approaches.

MASTL is the human ortholog of Greatwall kinase that facilitates mitotic entry, anaphase and cytokinesis

Greatwall (Gwl) was originally discovered in Drosophila as an essential kinase for correct chromosome condensation and mitotic progression. In Xenopus, Gwl may influence the positive-feedback loop that directs cyclin B1-Cdk1 activation and the mitotic state by inhibiting the phosphatase PP2A. Here, we describe the human orthologue of Gwl called microtubule-associated serine/threonine kinase-like (MASTL). We found that MASTL localizes to the nucleus in interphase and re-localizes in part to centrosomes in mitosis, when it is active. Cells strongly depleted of MASTL by RNAi delay in G2 phase and reveal slow chromosome condensation. MASTL RNAi cells that enter and progress through mitosis often fail to completely separate their sister chromatids in anaphase. This causes chromatin to be trapped in the cleavage furrow, which may lead to formation of 4N G1 cells by cytokinesis failure. Further, our experiments indicate that MASTL supports the phosphorylation state of mitotic phospho-proteins downstream of cyclin B1-Cdk1, including the APC/C. Cyclin B1 destruction is incomplete when mitotic cells that are strongly depleted of MASTL exit mitosis. We propose that MASTL enhances cyclin B1-Cdk1-dependent mitotic phosphorylation-events, directing mitotic entry, anaphase and cytokinesis in human cells.

Greatwall kinase protects mitotic phosphosites from barbarian phosphatases

The basic outline of the events controlling entry into mitosis has been clear for more than 20 y: the essential mitotic driver M-phase promoting factor [MPF; a cyclin-dependent kinase (CDK) composed of CDK1 and cyclin B] becomes rapidly activated and phosphorylates a wide variety of targets, creating mitotic phosphoproteins that contribute to nuclear membrane breakdown, chromosome condensation, and spindle assembly. But is this the entire story? The article in PNAS by Burgess et al. (1) adds to evidence accumulating over the last 2 y arguing that half the picture has been missing. The levels of mitotic phosphoproteins are governed not only by MPF but also by the specific phosphatases that remove MPF-catalyzed phosphorylations. Importantly, both entry into M phase and the maintenance of M phase require the operation of a pathway, mediated by a kinase called Greatwall (Gwl), that can inactivate these phosphatases.

Spindly/CCDC99 Is Required for Efficient Chromosome Congression and Mitotic Checkpoint Regulation

Spindly recruits a fraction of cytoplasmic dynein to kinetochores for poleward movement of chromosomes and control of mitotic checkpoint signaling. Here we show that human Spindly is a cell cycle-regulated mitotic phosphoprotein that interacts with the Rod/ZW10/Zwilch (RZZ) complex. The kinetochore levels of Spindly are regulated by microtubule attachment and biorientation induced tension. Deletion mutants lacking the N-terminal half of the protein (NDelta253), or the conserved Spindly box (DeltaSB), strongly localized to kinetochores and failed to respond to attachment or tension. In addition, these mutants prevented the removal of the RZZ complex and that of MAD2 from bioriented chromosomes and caused cells to arrest at metaphase, showing that RZZ-Spindly has to be removed from kinetochores to terminate mitotic checkpoint signaling. Depletion of Spindly by RNAi, however, caused cells to arrest in prometaphase because of a delay in microtubule attachment. Surprisingly, this defect was alleviated by codepletion of ZW10. Thus, Spindly is not only required for kinetochore localization of dynein but is a functional component of a mechanism that couples dynein-dependent poleward movement of chromosomes to their efficient attachment to microtubules.

CyclinB/cdk1‐mediated phosphorylation of the PR‐Set7 histone methyltransferase is required for its nuclear localization and stability during mitosis

We previously demonstrated that accumulation of PR‐Set7 and the histone modification that it creates, monomethyl‐H4K20, during G2/M are essential for mammalian cell cycle progression. However, it remains unclear how PR‐Set7 protein is regulated during mitosis. Based on recent findings, we hypothesized that PR‐Set7 is a mitotic phosphoprotein and that this phosphorylation event affects PR‐Set7 function during mitosis. Here we discovered that human PR‐Set7 is phosphorylated specifically by cyclinB/cdk1 and exclusively at G2/M. We found that PR‐Set7 phosphorylation occurs early during mitosis and that this event is required for the proper localization of PR‐Set7 within the nucleus. In addition, phosphorylation stabilizes PR‐Set7 protein levels by directly preventing its ubiquitination and proteasome‐mediated degradation. Importantly, impairment of PR‐Set7 function resulted in global chromatin decondensation, cell cycle and segregation defects. Collectively, our results reveal a novel mechanism by which cyclinB/cdk1‐mediated phosphorylation of PR‐Set7 functions to stabilize and direct this chromatin‐modifying enzyme to ensure normal cell division.

Dissecting the M Phase–specific Phosphorylation of Serine–Proline or Threonine–Proline Motifs

M phase induction in eukaryotic cell cycles is associated with a burst of protein phosphorylation, primarily at serine or threonine followed by proline (S/TP motif). The mitotic phosphoprotein antibody MPM-2 recognizes a significant subset of mitotically phosphorylated S/TP motifs; however, the required surrounding sequences of and the key kinases that phosphorylate these S/TP motifs remain to be determined. By mapping the mitotic MPM-2 epitopes in Xenopus Cdc25C and characterizing the mitotic MPM-2 epitope kinases in Xenopus oocytes and egg extracts, we have determined that phosphorylation of TP motifs that are surrounded by hydrophobic residues at both -1 and +1 positions plays a dominant role in M phase-associated burst of MPM-2 reactivity. Although mitotic Cdk and MAPK may phosphorylate subsets of these motifs that have a basic residue at the +2 position and a proline residue at the -2 position, respectively, the majority of these motifs that are preferentially phosphorylated in mitosis do not have these features. The M phase-associated burst of MPM-2 reactivity can be induced in Xenopus oocytes and egg extracts in the absence of MAPK or Cdc2 activity. These findings indicate that the M phase-associated burst of MPM-2 reactivity represents a novel type of protein phosphorylation in mitotic regulation.

Cep55 stabilization is required for normal execution of cytokinesis

Cep55 is a mitotic phosphoprotein that plays an important role in cytokinesis, the final stage of cell division during which physical separation of the two daughter cells is accomplished. We recently demonstrated that the peptidyl-prolyl isomerase Pin1 regulates this cell cycle event by enhancing the Plk1-dependent phosphorylation of Cep55. We show here that Cep55 is stabilized post-translationally during mitosis and that siRNA-mediated knockdown of Pin1 prevents this stabilization. Consistent with this, Cep55 is unstable in Pin1 knockout mouse embryonic fibroblasts. Moreover, mutation of the Pin1 binding sites in Cep55 reduces its stability during mitosis. Mutation of the Plk1 phosphorylation site also lowers Cep55 stability, whereas overexpression of Plk1 increases Cep55 levels, in keeping with Pin1 regulating Plk1-mediated phosphorylation of Cep55. Importantly, expression of wild-type Cep55 at levels similar to that of the phosphorylation mutants only partially reverts the cytokinesis defect induced by depletion of Cep55, indicating that inadequate levels of Cep55 prevent proper execution of cytokinesis. Taken together, these data provide more insight into the regulation of the final stages of cell division. As cytokinesis defects can cause chromosomal instability, knowledge about the processes that regulate normal cytokinesis adds to our understanding of events that lead to tumorigenesis.

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin-dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55 delta subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle--high in interphase and suppressed during mitosis. Depletion of PP2A-B55 delta (in interphase) from 'cycling' frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A-B55 delta was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A-B55 delta in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.

PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation

Loss of cell division cycle 2 (Cdc2, also known as Cdk1) activity after cyclin B degradation is necessary, but not sufficient, for mitotic exit. Proteins phosphorylated by Cdc2 and downstream mitotic kinases must be dephosphorylated. We report here that protein phosphatase-1 (PP1) is the main catalyst of mitotic phosphoprotein dephosphorylation. Suppression of PP1 during early mitosis is maintained through dual inhibition by Cdc2 phosphorylation and the binding of inhibitor-1. Protein kinase A (PKA) phosphorylates inhibitor-1, mediating binding to PP1. As Cdc2 levels drop after cyclin B degradation, auto-dephosphorylation of PP1 at its Cdc2 phosphorylation site (Thr 320) allows partial PP1 activation. This promotes PP1-regulated dephosphorylation at the activating site of inhibitor-1 (Thr 35) followed by dissociation of the inhibitor-1-PP1 complex and then full PP1 activation to promote mitotic exit. Thus, Cdc2 both phosphorylates multiple mitotic substrates and inhibits their PP1-mediated dephosphorylation.

Dab2 regulates clathrin assembly and cell spreading

The recruitment of clathrin to the membrane and its assembly into coated pits results from its interaction with endocytic adaptors and other regulatory proteins in the context of a specific lipid microenvironment. Dab2 (disabled 2) is a mitotic phosphoprotein and a monomeric adaptor for clathrin-mediated endocytosis. In the present study, we employed GFP (green fluorescent protein) fusion constructs of different isoforms and mutants of rat Dab2 and characterized their effect on the size, distribution and dynamics of clathrin assemblies. Enhanced levels of expression of the p82 isoform of Dab2 in COS7 cells induced enlarged clathrin assemblies at the plasma membrane. p82-clathrin assemblies, which concentrate additional endocytic proteins, such as AP2 (adaptor protein 2) and epsin, are dynamic structures in which both p82 and clathrin exchange actively between the membrane-bound and cytosolic sub-populations. The ability of p82 to induce enlarged clathrin assemblies is dependent on the presence of a functional PTB domain (phosphotyrosine-binding domain), on binding to clathrin and phospholipids, and on a newly identified and evolutionarily conserved poly-lysine stretch which precedes the PTB domain. These same molecular features are required for Dab2 to enhance the spreading of COS7 cells on fibronectin. The ability of the p82 isoform of Dab2 to enhance cell spreading was confirmed in both HeLa cells and HBL cells (human breast epithelial cells). COS7 cells expressing GFP-p82 and plated on to fibronectin concentrate the beta1 integrin into clathrin-p82 assemblies. Furthermore, during cell spreading, p82-clathrin assemblies concentrate at the site of the initial cell-matrix contact and are absent from regions of intense membrane ruffling. We propose a role for Dab2 and clathrin in integrin-mediated cell spreading.

Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis

The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cis-trans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo.

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.