Marcelo G. Kazanietz

Protein kinase C and other diacylglycerol effectors in cancer

Almost three decades after the discovery of protein kinase C (PKC), we still have only a partial understanding of how this family of serine/threonine kinases is involved in tumour promotion. PKC isozymes — effectors of diacylglycerol (DAG) and the main targets of phorbol-ester tumour promoters — have important roles in cell-cycle regulation, cellular survival, malignant transformation and apoptosis. How do PKC isozymes regulate these diverse cellular processes and what are their contributions to carcinogenesis? Moreover, what is the contribution of all phorbol-ester effectors, which include PKCs and small G-protein regulators? We now face the challenge of dissecting the relative contribution of each DAG signal to cancer progression.

New insights into the regulation of protein kinase C and novel phorbol ester receptors

Protein kinase C (PKC), a family of related serine‐threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid‐regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide‐dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein‐protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC‐independent pathways.—Ron, D., Kazanietz, M. G. New insights into the regulation of protein kinase C and novel phorbol ester receptors. FASEB J. 13, 1658–1676 (1999)

Crystal structure of the Cys2 activator-binding domain of protein kinase Cδ in complex with phorbol ester

Protein kinase Cs (PKCs) are a ubiquitous family of regulatory enzymes that associate with membranes and are activated by diacylglycerol or tumor-promoting agonists such as phorbol esters. The structure of the second activator-binding domain of PKC delta has been determined in complex with phorbol 13-acetate, which binds in a groove between two pulled-apart beta strands at the tip of the domain. The C3, C4, and C20 phorbol oxygens form hydrogen bonds with main-chain groups whose orientation is controlled by a set of highly conserved residues. Phorbol binding caps the groove and forms a contiguous hydrophobic surface covering one-third of the domain, explaining how the activator promotes insertion of PKC into membranes.

Protein Kinase C Promotes Apoptosis in LNCaP Prostate Cancer Cells through Activation of p38 MAPK and Inhibition of the Akt Survival Pathway

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKCα and the novel PKCδ isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574–7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645–655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKCα in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Arachidonic Acid in Platelet Microparticles Up-regulates Cyclooxygenase-2-dependent Prostaglandin Formation via a Protein Kinase C/Mitogen-activated Protein Kinase-dependent Pathway

Activation of platelets results in shedding of membrane microparticles (MP) with potentially bioactive properties. Platelet MP modulate platelet, monocyte, and vascular endothelial cell function, both by direct effects of MP arachidonic acid (AA) and by its metabolism to bioactive prostanoids. We have previously reported that platelet MP induce expression of cyclooxygenase (COX)-2 and prostacyclin production in monocytes and endothelial cells. To elucidate further the molecular mechanisms that underlie MP-induced up-regulation of COX-2 expression, we investigated the response of a human monocytoid (U-937) cell line to platelet MP stimulation. In U-937 cells, MP-induced COX-2 expression and eicosanoid formation is prevented by pharmacological inhibitors of protein kinase C (PKC), PI 3-kinase, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, and p38 kinase. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 also blocked MP-induced p42/p44 MAPK, p38, and JNK1 phosphorylation. Conversely, platelet MP stimulation of U-937 cells results in direct activation of PKC, p42/p44 MAPK, p38 kinase, and c-Jun N-terminal kinase (JNK) as well as activation of the transcription factors c-Jun and Elk-1. However, MP failed to activate the cAMP response element. Activation of U-937 cells by MP induces translocation of classical (PKCβ), novel (PKCδ) and atypical (PKCζ and PKCλ) isozymes of PKC from the cytosol to the membrane, with concomitant activation of downstream MAPK. While MP-induced activation of p42/p44 MAPK and p38 kinase is transient, a sustained activation of JNK1 was observed. Although PKC activation is required for MP-induced p42/p44 MAPK, activation of the stress kinases p38 and JNK1 was PKC-independent. The fatty acid fraction of the MP accounted for these effects, which were mimicked by MP AA. Rather than acting directly via nuclear receptors, MP AA activates COX-2-dependent prostaglandin production by a PKC/p42/p44 MAPK/p38 kinase-sensitive pathway in which PI 3-kinase plays a significant role. MP AA also stimulates transcriptional activation of COX-2 as well as c-Jun and Elk-1.

Involvement of Protein Kinase C δ (PKCδ) in Phorbol Ester-induced Apoptosis in LNCaP Prostate Cancer Cells LACK OF PROTEOLYTIC CLEAVAGE OF PKCδ

Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKCδ, we used a replication-deficient adenovirus (PKCδAdV), which allowed for a tightly controlled expression of PKCδ in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKCδAdV. Overexpression of PKCδ markedly enhanced the apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells. PKCδ-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKCδ-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKCδ is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKCδ, supporting the concept that PKCδ plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.

Eyes wide shut: protein kinase C isozymes are not the only receptors for the phorbol ester tumor promoters.

In addition to the well‐characterized interaction with classical and novel protein kinase C (PKC) isozymes, the phorbol ester tumor promoters bind to other receptors lacking kinase activity. Among these novel phorbol ester receptors, two families of proteins may play a role in the regulation of cell growth and malignant transformation: chimaerins and ras guanyl–releasing protein (ras‐GRP). These proteins possess a single copy of the C1 domain that is involved in binding of phorbol esters and the lipid second messenger diacylglycerol. Four isoforms of chimaerins (α1‐, α2‐, β1‐, and β2‐chimaerins) have been isolated to‐date, all of them possessing GTPase‐activating protein activity for Rac, a small GTP‐binding protein that controls actin cytoskeleton organization, cell‐cycle progression, adhesion, and migration. Ras‐GRP is a guanine nucleotide exchange factor for ras and promotes malignant transformation in fibroblasts in a phorbol ester–dependent manner. The C1 domain in Ras‐GRP may, therefore, have a dominant role in Ras‐GRP activation and is essential for phorbol ester–dependent activation of downstream effectors of ras, i.e., the mitogen‐activated protein kinase cascade. Thus, a novel concept emerges in which phorbol esters may exert cellular responses through pathways not involving phorbol ester–responsive PKC isozymes. The discovery of “nonPKC” phorbol ester receptors adds an additional level of complexity to the understanding of phorbol ester effects and the molecular mechanisms of carcinogenesis. Mol. Carcinog. 28:5–11, 2000.

Residues in the second cysteine-rich region of protein kinase C delta relevant to phorbol ester binding as revealed by site-directed mutagenesis.

Phorbol esters bind with high affinity to protein kinase C (PKC) isozymes as well as to two novel receptors, n-chimaerin and Unc-13. The cysteine-rich regions present in these proteins were identified as the binding sites for the phorbol ester tumor promoters and the lipophilic second messenger sn-diacylglycerol. A 50-amino-acid peptide comprising the second cysteine-rich region of PKC , expressed in Escherichia coli as a glutathione S-transferase (GST)-fusion protein, bound phorbol 12,13-dibutyrate (PDBu) with high affinity (K =0.8 nM). Using the cDNA of that cysteine-rich region as a template, a series of 37 point mutations was generated by site-directed mutagenesis, and the mutated proteins were analyzed quantitatively for binding of [H]PDBu and, as appropriate, for binding of the ultrapotent analog [H]bryostatin 1. Mutants displayed one of three patterns of behavior: phorbol ester binding was completely abolished, binding affinity was reduced, or binding was not significantly modified. As expected, five of the six cysteines as well as the two histidines involved in Zn coordination are critical for the interaction of the protein with the phorbol esters. In addition, mutations in several positions, including phenylalanine 3, tyrosine 8, proline 11, leucines 20, 21, and 24, tryptophan 21, glutamine 27, and valine 38 drastically reduced the interaction with the ligands. The effect of these mutations can be rationalized from the three-dimensional (NMR) structure of the cysteine-rich region. In particular, the C-terminal portion of the protein does not appear to be essential, and the loop comprising amino acids 20 to 28 is implicated in the binding activity.

Hedgehog proteins activate pro-angiogenic responses in endothelial cells through non-canonical signaling pathways.

The Hedgehog (Hh) pathway orchestrates developmental and homeostatic angiogenesis. The three Hh isoforms- Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh)-signal through Patched-1 (PTCH1) and Smoothened (SMO), to activate the Gli transcription factors with a characteristic rank of potency (Shh>>Ihh>Dhh). To dissect the mechanisms through which Hh proteins promote angiogenesis, we analyzed processes inherent to vessel formation in endothelial cells. We found that none of the Hh ligands were able to induce Gli-target genes in human umbilical vein (HUVEC) or human cardiac microvascular endothelial cells (HMVEC), suggesting that endothelial cells do not respond to Hh through the canonical pathway. However, our results show that the three Hh proteins promote endothelial cell tubulogenesis in 3D cultures in a SMO- and Gi protein-dependent manner. Consistent with the required cytoskeletal rearrangement for tubulogenesis, Shh, Ihh, and Dhh all stimulated the small GTPase RhoA and the formation of actin stress fibers. This effect, which was mediated by SMO, Gi proteins, and Rac1, defines a new non-canonical Hh pathway. In addition to regulating the actin cytoskeleton, the Hh ligands promoted survival through inhibition of the pro-apoptotic effect of PTCH1 in a SMO-independent manner. Altogether, our results support the existence of Gli-independent Hh responses in endothelial cells that regulate tubulogenesis and apoptosis. The identification of novel non-canonical responses elicited by Hh proteins in endothelial cells highlights the complexity of the Hh signaling pathway and reveals striking differences in ligand strength for transcriptional and non-transcriptional responses.

Identification of the Rac-GEF P-Rex1 as an Essential Mediator of ErbB Signaling in Breast Cancer

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.