Feasibility study of a multimodal prehabilitation programme in women receiving neoadjuvant therapy for breast cancer in a major cancer hospital: a protocol.

INTRODUCTION: Neoadjuvant therapy has become a standard treatment for patients with stage II/III HER2 positive and triple negative breast cancer, and in well-selected patients with locally advanced and borderline resectable high risk, luminal B breast cancer. Side effects of neoadjuvant therapy, such as fatigue, cardiotoxicity, neurotoxicity, anxiety, insomnia, vasomotor symptoms, gastrointestinal disturbance as well as a raft of immune-related adverse events, may impact treatment tolerance, long-term outcomes, and quality of life. Providing early supportive care prior to surgery (typically termed 'prehabilitation') may mitigate these side effects and improve quality of life.During our codesign of the intervention, consumers and healthcare professionals expressed desire for a programme that 'packaged' care, was easy to access, and was embedded in their care pathway. We hypothesise that a multimodal supportive care programme including exercise and complementary therapies, underpinned by behavioural change theory will improve self-efficacy, quality of life, readiness for surgery and any additional treatment for women with breast cancer. We seek to explore cardiometabolic, residual cancer burden and surgical outcomes, along with chemotherapy completion (relative dose intensity). This article describes the protocol for a feasibility study of a multimodal prehabilitation programme. METHODS AND ANALYSIS: This is a prospective, mixed-method, feasibility study of a multi-modal programme in a hospital setting for 20-30 women with breast cancer receiving neoadjuvant therapy. Primary outcomes are recruitment rate, retention rate, adherence and acceptability. Secondary outcomes include patient reported outcome measures (PROMs), surgical outcomes, length of stay, satisfaction with surgery, chemotherapy completion rates, changes in metabolic markers and adverse events. Interviews and focus groups to understand the experience with prehabilitation and different factors that may affect feasibility of the intervention . The output of this study will be a codesigned, evidence-informed intervention assessed for feasibility and acceptability by women with breast cancer and the healthcare professionals that care for them. ETHICS AND DISSEMINATION: The study received ethics approval from the St Vincents Hospital HREC (HREC/2021/ETH12198). Trial results will be communicated to participants, healthcare professionals, and the public via publication and conferences. TRIAL REGISTRATION NUMBER: ACTRN12622000584730.

MAPKAP Kinase-2 phosphorylation of PABPC1 controls its interaction with 14-3-3 proteins after DNA damage: A combined kinase and protein array approach.

14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

High-Content Imaging of Unbiased Chemical Perturbations Reveals that the Phenotypic Plasticity of the Actin Cytoskeleton Is Constrained.

Although F-actin has a large number of binding partners and regulators, the number of phenotypic states available to the actin cytoskeleton is unknown. Here, we quantified 74 features defining filamentous actin (F-actin) and cellular morphology in >25 million cells after treatment with a library of 114,400 structurally diverse compounds. After reducing the dimensionality of these data, only ∼25 recurrent F-actin phenotypes emerged, each defined by distinct quantitative features that could be machine learned. We identified 2,003 unknown compounds as inducers of actin-related phenotypes, including two that directly bind the focal adhesion protein, talin. Moreover, we observed that compounds with distinct molecular mechanisms could induce equivalent phenotypes and that initially divergent cellular responses could converge over time. These findings suggest a conceptual parallel between the actin cytoskeleton and gene regulatory networks, where the theoretical plasticity of interactions is nearly infinite, yet phenotypes in vivo are constrained into a limited subset of practicable configurations.

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac-mediated multicellular invasion.

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion.

On-target action of anti-tropomyosin drugs regulates glucose metabolism.

The development of novel small molecule inhibitors of the cancer-associated tropomyosin 3.1 (Tpm3.1) provides the ability to examine the metabolic function of specific actin filament populations. We have determined the ability of these anti-Tpm (ATM) compounds to regulate glucose metabolism in mice. Acute treatment (1 h) of wild-type (WT) mice with the compounds (TR100 and ATM1001) led to a decrease in glucose clearance due mainly to suppression of glucose-stimulated insulin secretion (GSIS) from the pancreatic islets. The impact of the drugs on GSIS was significantly less in Tpm3.1 knock out (KO) mice indicating that the drug action is on-target. Experiments in MIN6 ß-cells indicated that the inhibition of GSIS by the drugs was due to disruption to the cortical actin cytoskeleton. The impact of the drugs on insulin-stimulated glucose uptake (ISGU) was also examined in skeletal muscle ex vivo. In the absence of drug, ISGU was decreased in KO compared to WT muscle, confirming a role of Tpm3.1 in glucose uptake. Both compounds suppressed ISGU in WT muscle, but in the KO muscle there was little impact of the drugs. Collectively, this data indicates that the ATM drugs affect glucose metabolism in vivo by inhibiting Tpm3.1's function with few off-target effects.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

Tumor suppressor tropomyosin Tpm2.1 regulates sensitivity to apoptosis beyond anoikis characterized by changes in the levels of intrinsic apoptosis proteins.

The actin cytoskeleton is a polymer system that acts both as a sensor and mediator of apoptosis. Tropomyosins (Tpm) are a family of actin binding proteins that form co-polymers with actin and diversify actin filament function. Previous studies have shown that elevated expression of the tropomyosin isoform Tpm2.1 sensitized cells to apoptosis induced by cell detachment (anoikis) via an unknown mechanism. It is not yet known whether Tpm2.1 or other tropomyosin isoforms regulate sensitivity to apoptosis beyond anoikis. In this study, rat neuroepithelial cells overexpressing specific tropomyosin isoforms (Tpm1.7, Tpm2.1, Tpm3.1, and Tpm4.2) were screened for sensitivity to different classes of apoptotic stimuli, including both cytoskeletal and non-cytoskeletal targeting compounds. Results showed that elevated expression of tropomyosins in general inhibited apoptosis sensitivity to different stimuli. However, Tpm2.1 overexpression consistently enhanced sensitivity to anoikis as well as apoptosis induced by the actin targeting drug jasplakinolide (JASP). In contrast the cancer-associated isoform Tpm3.1 inhibited the induction of apoptosis by a range of agents. Treatment of Tpm2.1 overexpressing cells with JASP was accompanied by enhanced sensitivity to mitochondrial depolarization, a hallmark of intrinsic apoptosis. Moreover, Tpm2.1 overexpressing cells showed elevated levels of the apoptosis proteins Bak (proapoptotic), Mcl-1 (prosurvival), Bcl-2 (prosurvival) and phosphorylated p53 (Ser392). Finally, JASP treatment of Tpm2.1 cells caused significantly reduced Mcl-1, Bcl-2 and p53 (Ser392) levels relative to control cells. We therefore propose that Tpm2.1 regulates sensitivity to apoptosis beyond the scope of anoikis by modulating the expression of key intrinsic apoptosis proteins which primes the cell for death.

A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics.

The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development.

Hypoxia alters the recruitment of tropomyosins into the actin stress fibres of neuroblastoma cells.

BACKGROUND: Neuroblastoma is the most common extracranial solid tumor of childhood. The heterogeneous microenvironment of solid tumors contains hypoxic regions associated with poor prognosis and chemoresistance. Hypoxia implicates the actin cytoskeleton through its essential roles in motility, invasion and proliferation. However, hypoxia-induced changes in the actin cytoskeleton have only recently been observed in human cells. Tropomyosins are key regulators of the actin cytoskeleton and we hypothesized that tropomyosins may mediate hypoxic phenotypes. METHODS: Neuroblastoma (SH-EP) cells were incubated ± hypoxia (1 % O2, 5 % CO2) for up to 144 h, before examining the cytoskeleton by confocal microscopy and Western blotting. RESULTS: Hypoxic cells were characterized by a more organized actin cytoskeleton and a reduced ability to degrade gelatin substrates. Hypoxia significantly increased mean actin filament bundle width (72 h) and actin filament length (72-96 h). This correlated with increased hypoxic expression and filamentous organization of stabilizing tropomyosins Tm1 and Tm2. However, isoform specific changes in tropomyosin expression were more evident at 96 h. CONCLUSIONS: This study demonstrates hypoxia-induced changes in the recruitment of high molecular weight tropomyosins into the actin stress fibres of a human cancer. While hypoxia induced clear changes in actin organization compared with parallel normoxic cultures of neuroblastoma, the precise role of tropomyosins in this hypoxic actin reorganization remains to be determined.

Cell elasticity is regulated by the tropomyosin isoform composition of the actin cytoskeleton.

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.

Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments.

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor-stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

Validation of an algorithm to quantify changes in actin cytoskeletal organization.

The actin cytoskeleton plays an important role in most, if not all, processes necessary for cell survival. Given the fundamental role that the actin cytoskeleton plays in the progression of cancer, it is an ideal target for chemotherapy. Although it is possible to image the actin cytoskeleton in a high-throughput manner, there is currently no validated method to quantify changes in the cytoskeleton in the same capacity, which makes research into its organization and the development of anticytoskeletal drugs difficult. We have validated the use of a linear feature detection algorithm, allowing us to measure changes in actin filament organization. Its ability to quantify changes associated with cytoskeletal disruption will make it a valuable tool in the development of compounds that target the cytoskeleton in cancer. Our results show that this algorithm can quantify cytoskeletal changes in a cell-based system after addition of both well-established and novel anticytoskeletal agents using either fluorescence microscopy or a high-content imaging approach. This novel method gives us the potential to screen compounds in a high-throughput manner for cancer and other diseases in which the cytoskeleton plays a key role.

A novel class of anticancer compounds targets the actin cytoskeleton in tumor cells.

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

The actin cytoskeleton as a sensor and mediator of apoptosis.

Apoptosis is an important biological process required for the removal of unwanted or damaged cells. Mounting evidence implicates the actin cytoskeleton as both a sensor and mediator of apoptosis. Studies also suggest that actin binding proteins (ABPs) significantly contribute to apoptosis and that actin dynamics play a key role in regulating apoptosis signaling. Changes in the organization of the actin cytoskeleton has been attributed to the process of malignant transformation and it is hypothesized that remodeling of the actin cytoskeleton may enable tumor cells to evade normal apoptotic signaling. This review aims to illuminate the role of the actin cytoskeleton in apoptosis by systematically analyzing how actin and ABPs regulate different apoptosis pathways and to also highlight the potential for developing novel compounds that target tumor-specific actin filaments.

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments.

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.

Tropomyosin isoform expression regulates the transition of adhesions to determine cell speed and direction.

The balance of transition between distinct adhesion types contributes to the regulation of mesenchymal cell migration, and the characteristic association of adhesions with actin filaments led us to question the role of actin filament-associating proteins in the transition between adhesive states. Tropomyosin isoform association with actin filaments imparts distinct filament structures, and we have thus investigated the role for tropomyosins in determining the formation of distinct adhesion structures. Using combinations of overexpression, knockdown, and knockout approaches, we establish that Tm5NM1 preferentially stabilizes focal adhesions and drives the transition to fibrillar adhesions via stabilization of actin filaments. Moreover, our data suggest that the expression of Tm5NM1 is a critical determinant of paxillin phosphorylation, a signaling event that is necessary for focal adhesion disassembly. Thus, we propose that Tm5NM1 can regulate the feedback loop between focal adhesion disassembly and focal complex formation at the leading edge that is required for productive and directed cell movement.

New approaches to targeting the actin cytoskeleton for chemotherapy.

The actin cytoskeleton is indispensable for normal cellular function. In particular, several actin-based structures coordinate cellular motility, a process hijacked by tumor cells in order to facilitate their propagation to distant sites. The actin cytoskeleton, therefore, represents a point for chemotherapeutic intervention. The challenge in disrupting the actin cytoskeleton is in preserving actin-driven contraction of cardiac and skeletal muscle. By targeting actin-binding proteins with altered expression in malignancy, it may be possible to achieve tumor-specific toxicity. A number of actin-binding proteins act cooperatively and synergistically to regulate actin structures required for motility. The actin cytoskeleton is characterized by a significant degree of plasticity. Targeting specific actin-binding proteins for chemotherapy will only be successful if no other compensatory mechanisms exist.

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility.

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.

Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C beta II and coincides with the onset of stimulated degranulation of RBL-2H3 mast cells.

Dynamic remodeling of the actinomyosin cytoskeleton is integral to many biological processes. It is regulated, in part, by myosin phosphorylation. Nonmuscle myosin H chain IIA is phosphorylated by protein kinase C (PKC) on Ser(1917). Our aim was to determine the PKC isoform specificity of this phosphorylation event and to evaluate its potential role in regulated secretion. Using an Ab against the phosphorylated form of Ser(1917), we show that this site is not phosphorylated in unstimulated RBL-2H3 mast cells. The physiological stimulus, Ag, or the pharmacological activators, PMA plus A23187, induced Ser(1917) phosphorylation with a time course coincident with the onset of granule mediator secretion. Dephosphorylation at this site occurred as Ag-stimulated secretion declined from its peak, but dephosphorylation was delayed in cells activated with PMA plus A23187. Phosphate incorporation was also enhanced by PMA alone and by inhibition of protein phosphatase 2A. Gö6976, an inhibitor of conventional PKC isoforms, abolished secretion and Ser(1917) phosphorylation with similar dose dependencies consistent with involvement of either PKCalpha or PKCbeta. Phorbol ester-stimulated Ser(1917) phosphorylation was reconstituted in HEK-293 cells (which lack endogenous PKCbeta) by overexpression of both wild-type and constitutively active PKCbetaII but not the corresponding PKCbetaI or PKCalpha constructs. A similar selectivity for PKCbetaII overexpression was also observed in MIN6 insulinoma cells infected with recombinant PKC wild-type adenoviruses. Our results implicate PKC-dependent phosphorylation of myosin H chain IIA in the regulation of secretion in mast cells and suggest that Ser(1917) phosphorylation might be a marker of PKCbetaII activation in diverse cell types.