Introduction: Protein Kinase C
Cellular proliferation is a highly complex process driven by specific signals that stimulate cells to replicate and generate “daughter” cells, or copies of themselves. Among the various tissues in the body, there are those that are subject to continuous renewal (e.g., bone marrow, skin, internal mucosa, nails) and, therefore, have a high proliferative rate. Cell growth is induced by any substance that allows the passage of signals from the cell membrane through the cytoplasm and into the nucleus, where it triggers gene transcription mechanisms.
A signaling pathway typically involved in cell replication is that of calcium/lipid-dependent protein kinase (PKC), which recognizes the lipid diacyl-glycerol, or DAG, as its activating ligand. PKCs are a family of kinases that can be activated by growth factors, hormones, neurotransmitters, and even some natural compounds. They are divided into classical (alpha, beta, gamma), which require calcium, DAG and phosphatidyl serine (PhS); atypical (zeta, iota, lambda), which require only PhS and are regulated by interactions with other partner proteins; and finally, novel isoforms (delta, epsilon, eta, theta), which are regulated by DAG and related lipids (PhS, phosphatidic acid) but without the intervention of calcium.
The gamma isoform is the only one also activated by arachidonic acid released by the hydrolysis of membrane lipids by phospholipase α2 (PLA2). Classical PKCs are also activated by natural ligands called phorbolic esters, found in some irritating plants. Because they are non-degradable, their activation of PKC is persistent, making them very useful in studying cell proliferation in oncology. Phorbol esters, in fact, are recognized as “tumor promoters”, although not directly carcinogenic.
But since the 1980s and 1990s, when the regulation of PKCs by DAG and phorbols was discovered, other DAG targets have been identified that could explain certain cellular actions of DAG in certain cellular contexts. PKCs, in fact, are not uniformly distributed throughout all cell types in the body, so certain actions that cannot be explained by the presence of classical DAG-sensitive PKCs must necessarily be attributed to the binding of this second messenger to other target proteins.
Additional Molecular Targets of DAG
1) Protein Kinase D (PKD1/2/3; formerly called PKCμ for PKD1)
It is a Ser/Thr kinase with two C1 domains (which bind free lipids) that mediate recruitment/activation in response to DAG (with important functions in membrane trafficking, the Golgi compartment, secretion, migration, and survival).
2) RasGRP (Ras guanine nucleotide–releasing proteins; isoforms 1/3/4)
They are exchange factors (GEFs) for Ras/Rap: DAG promotes membrane translocation via the C1 domain and connects the PLC→DAG phospholipase pathway to the Ras/MAPK signaling pathway, particularly in lymphocytes. Note: Some isoforms have C1 domains that are less responsive to DAG than others (differences between members).
3) Chimaerins (α- and β-chimaerin; CHN1/CHN2)
Historically, these are among the first “non-PKC” substrates recognized as receptors for DAG or phorbol esters. They are Rac-GAPs: DAG binding to C1 promotes membrane association and cytoskeletal/motility regulation because GAP proteins deactivate their activity by promoting hydrolysis of GTP bound to the Rac1 protein.
4) Munc13 / UNC13 (UNC13A/B/C)
Key proteins in synaptic vesicle priming (and, in some contexts, regulated secretion): DAG formed in the membrane modulates their activity via the C1 domain, increasing the likelihood of release.
5) Diacylglycerol kinases (DAGK; especially DGKγ)
Here the relationship is “special”: DAGKs consume DAG, converting it to phosphatidic acid (PhA), then suppress/modulate the DAG signal and generate a second lipid signal (PhA) that has its downstream substrates (protein kinases, ion channels). However, some isoforms are classified as non-PKC receptors due to their C1 domains and regulation by DAG/analogs.
6) MRCK (Myotonic dystrophy kinase-related Cdc42-binding kinases; MRCKα/β/γ)
ROCK-1-like kinases with membrane-binding modules: it has been documented that the C1 domain of MRCK can interact with DAG, influencing cytoplasmic actomyosin contractility, cell polarity, and migration.
7) TRPC3/6/7 ion channels
These “canonical TRP” cation channels are a classic example of a target directly activated by DAG in a PKC-independent manner (membrane-level integration mechanism). They allow ions (sodium, calcium) to pass through the cell membrane, generating electrical signals that transmit information to the brain, influencing pain, fever, cough, cold perception, and other physiological functions.
How are alternative targets translated into specific contexts?
- A) Cancer cells
In cancer cells, DAG is often generated downstream of hyperactivated receptors (RTK/GPCR) via PLC-gamma phospholipase, creating membrane platforms (lipid rafts) that recruit DAG-sensitive effectors. A review of the cancer context highlights that DAG conveys proliferation, survival, and motility signals through PKC but also through non-PKC effectors such as PKD and regulators of small GTPases (e.g., RasGRP).
1) DAG → PKD (Protein Kinase D) axis: proliferation, transcription, migration
PKDs (PKD1/2/3) are kinases with C1 domains that respond to DAG and are involved in many tumor-promoting functions (proliferation, survival, migration, invasion, transcriptional programs, and secretion of pro-tumor factors), with roles that can be pro- or anti-tumor depending on the context and isoform.
2) DAG → RasGRP → MAPK axis: cell cycle entry
RasGRPs are GEFs with a C1 domain: DAG promotes their recruitment to the membrane and activation of the Ras/MAPK cascade. In some tumors, this axis is explicitly engaged to support proliferative signals: for example, RasGRP3 has been described as a mediator of MAPK activation in a specific oncogenic context (uveal melanoma associated with G-alphaQ/11 mutations).
3) DAG → TRPC axis: Ca²⁺ as a mitogenic co-signal
A key point (often overlooked) is that some TRPC channels are directly activated by DAG (a PKC-independent mechanism), increasing the influx of Ca²⁺ ions and fueling proliferative and cell cycle progression programs. The DAG→TRPC3/6 link is a classic and well-established finding; in oncology, TRPC6 has been linked to proliferation and progression in several tumors (e.g., glioma; breast cancer lines).
4) Oncology “Control Valve”: DAGKs
DAGKs suppress/modulate DAG signaling by converting it to phosphatidic acid (PA), which in turn is bioactive. In oncology, many DAGKs are intertwined with nodes such as Akt/mTOR and MAPK/ERK, influencing survival, angiogenesis, and migration; DAGKα, in particular, has been proposed as a therapeutic target in cancer.
In oncology summary: when DAG production is persistent (continuous oncogenic stimuli, remodeling of PLC/DGK/lipid enzymes, “stable” membrane microdomains), the functional output tends to shift toward mitogenic and pro-survival signals (MAPKs, transcriptional programs, Ca²⁺ entry, pro-tumor secretion).
- B) Neuronal Cells
Neuron: DAG as a Director of Synaptic Events
In neurons, DAG often arises downstream of metabotropic receptors and phospholipase PLCβ (but also in other contexts) and accumulates in highly localized microdomains (especially presynaptic or perisynaptic), with short lifespans and strong enzymatic control. Here, DAG does not “drive” cell division: instead, it orchestrates release probability, vesicle dynamics and neuroplasticity phenomena.
1) DAG → Munc13: Vesicle Priming and Release Probability
Munc13-1 (UNC13 family) is a pivotal presynaptic effector: it possesses a C1 domain that binds DAG (and phorbol esters), and its activation is linked to the reduction of the energy barrier for vesicle fusion, increasing release efficiency and modulating synaptic transmission. This role is supported by experimental work directly linking Munc13-1 C1 activation to changes in synaptic parameters and fusion mechanisms.
2) DAG as a “retrograde messenger precursor”
A crucial difference compared to tumor cells is that in the brain, DAG is not only a second messenger: it is also a substrate for the synthesis of 2-arachidonoyl-glycerol (2-AG) via DAG lipase (DAGLα). 2-AG is one of the best-characterized retrograde messengers: produced postsynaptically, it acts on presynaptic cannabinoid receptors and mediates forms of retrograde suppression of transmission. Genetic/functional evidence points to a central role for DAGLα in this pathway.
3) DAG ↔ DAGK at synapses: fine “sculpture” of plasticity
In neurons, DGK and DAG metabolism are considered organizational components of synaptic plasticity: by modulating the duration and localization of DAG (and the production of PhA), they contribute to balancing LTP/LTD processes and receptor/vesicle trafficking. Phosphatidic acid, in fact, regulates the Raf-MEK pathway and the activation of the mTOR protein complex, necessary for the synthesis of cellular proteins induced by various processes.
4) DAG → TRPC3/6/7: excitability and local Ca2⁺ signaling
TRPC3/6/7 can also be activated by DAG in neurons, contributing to local Ca2⁺ signals that influence excitability and synaptic integration. Furthermore, there is a feedback control (e.g., via PKC) on some TRPCs that limits excessive activation.
In neuronal summary: here the DAG is “encoded” as a fast and localized impulse that regulates synaptic machinery (Munc13), or as a slow regulator via plasticity pathways (including the production of 2-AG), not as a clonal growth signal.
- Edited by Dr. Gianfrancesco Cormaci, PhD, specialist in Clinical Biochemistry.
Scientific references
Cooke M et al. Sci Signal. 2022 Apr; 15(729):eabo0264.
Roy A et al. Biochim Biophys Acta. 2017; 1868(1):283.
Purow B. Clin Cancer Res. 2015; 21(22):5008–5012.
Tanimura A et al. Neuron. 2010 Feb 11; 65(3):320-327.
Basu J et al. J Neurosci. 2007 Jan; 27(5):1200–1210.
Brose N, Rosemund C et al. J Cell Sci. 2002; 115:4399.
Hofmann T et al. Nature. 1999 Jan; 397(6716):259-63.
