Spirulina and RAS/RAF/MAPK Signalling.

RAS GTPase Cycle: GEF, GAP, and Effectors

RAS proteins (HRAS, KRAS4A/4B, NRAS; 21 kDa; G-domain; GTP/GDP binding; Mg²&sup+;-coordination; intrinsic GTPase Kcat ~0.02/min) cycle between GDP-bound inactive and GTP-bound active states. Growth factor receptor activation → Grb2/SOS (SOS1/2 GEF; CDC25 domain; catalyses GDP→GTP exchange; Kcat ~50/min) → RAS-GTP → effector engagement. RAS-GAPs (GTPase-activating proteins: NF1/neurofibromin, RASA1/p120GAP, RASA2, DAB2IP) accelerate intrinsic GTPase 10&sup5;-fold by inserting catalytic arginine “finger” (Arg789 in RASA1) into RAS active site, stabilising the transition state. RAS-GTP activates: RAF (CRAF/RAF1; BRAF; ARAF) via RBD (RAS-binding domain; Arg89 Zn-coordinated; BRAF Val600Glu gain-of-function mutation bypasses RAS-dependence); PI3Kα p110α RBD (KRAS4B CAAX-membrane PI3K co-clustering); RALGDS (RAL GEF); TIAM1 (RAC1 GEF). KRAS4B is preferentially membrane-associated via a polybasic region and farnesyl modification at Cys185 (farnesyltransferase target).

RAF–MEK–ERK Cascade

RAS-GTP recruits CRAF to the plasma membrane (CRAF RBD Kd ~1–10 nM); 14-3-3 dissociates from CRAF Ser259/621 → CRAF dephosphorylation at Ser259 by PP2A → CRAF active. CRAF forms homo- or heterodimers (with BRAF) via their CRD and kinase domain interfaces (the KSR1 scaffold facilitates complex assembly). CRAF phosphorylates MEK1 Ser218/222 and MEK2 Ser222/226 → MEK1/2 active → ERK1 Thr202/Tyr204 and ERK2 Thr185/Tyr187 dual phosphorylation (TEY motif; MEK is the only known ERK kinase). pERK dimerises, translocates to nucleus, and phosphorylates transcription factors: ELK1 (SRE/CArG box; immediate early genes FOS/JUN/EGR1); c-Myc Ser62 (stabilisation); CREB Ser133 (CRE-driven genes); RSK1/2/3/4 (cytoplasmic; phosphorylates S6 Ser235/236; CREB Ser133; BAD Ser112; IRS-1 Ser302). DUSPs (dual-specificity phosphatases; DUSP1/MKP-1 nuclear; DUSP6/MKP-3 cytoplasmic; DUSP4/MKP-2) dephosphorylate both pThr and pTyr on ERK (Kcat ~100/min), providing rapid signal termination; DUSP1/6 are Nrf2 targets (ARE-containing promoters).

NF-κB–RAS–ERK Crosstalk

NF-κB and ERK engage in crosstalk: NF-κB drives expression of EGFR ligands (amphiregulin, HB-EGF) → autocrine EGFR → SOS→RAS→ERK. ERK phosphorylates IKKβ Ser177 (activating) in some contexts, providing RAS→NF-κB link; ERK also phosphorylates and stabilises c-Jun/c-Fos → AP-1→NF-κB co-regulation of inflammatory genes. TNF-α→TRAF2→ASK1/MEKK1→MKK4→JNK or MKK3→p38 (parallel stress kinase pathways; ERK and JNK/p38 often have opposing roles: ERK survival; JNK/p38 apoptosis/inflammation).

Spirulina’s Mechanistic Actions

• NF-κB ↓ → EGFR ligands ↓ → RAS-ERK ↓ (inflammatory context): PCB→NF-κB↓→amphiregulin/HB-EGF ↓ 20–35%→autocrine EGFR→SOS→RAS-GTP ↓→ERK1/2 pTyr204 ↓ 20–30% in LPS/TNF-stimulated cells. Inflammatory ERK suppressed without eliminating growth-factor-induced physiological ERK.
• Nrf2 → DUSP1/DUSP6 → ERK dephosphorylation ↑: Nrf2→DUSP1 ↑ 20–35% + DUSP6 ↑ 15–25% → pERK Thr202/Tyr204 half-life ↓ 30–40% in oxidative stress models→ERK amplitude ↓; similarly DUSP1 dephosphorylates JNK Thr183/Tyr185 and p38 Thr180/Tyr182 → multi-MAPK dampening.
• RAS-GAP (NF1/RASA1) Cys protection: NF1 and RASA1 contain critical Cys residues near their RAS-GAP active site; oxidation → S-glutathionylation → impaired Arg-finger insertion → reduced RAS-GTP hydrolysis → RAS hyperactivation. PCB→ROS↓→NF1/RASA1 Cys protected→RAS-GAP activity maintained→RAS-GTP levels normalised.
• AMPK → B-RAF Ser365 inhibitory phosphorylation: AMPK directly phosphorylates B-RAF Ser365 → B-RAF/CRAF heterodimerisation ↓→ERK output ↓ in growth-factor-over-stimulated contexts (nutrient surplus); ERK1/2 pThr202 ↓ 15–25% in adipose/liver models of over-nutrition.
• RSK→CREB Ser133: Spirulina’s context-dependent ERK modulation affects RSK→CREB Ser133→CRE-driven gene expression; in neuronal models, BDNF-driven ERK→RSK→CREB survival signalling is preserved (physiological ERK maintained; toxic inflammatory ERK suppressed).

Clinical Correlates and Dosing

Animal models: spirulina (50–200 mg/kg) suppresses LPS-induced pERK1/2 25–35% in macrophages and liver; tumour models (HCC, CRC) show B-RAF/ERK pathway ↓ correlating with anti-proliferative effects. In neuronal protection models, ERK survival signalling preserved while JNK/p38 stress kinases suppressed (dual beneficial profile). Human: pERK is not routinely measured in clinical spirulina trials; indirect: IL-6 (ERK-upstream/downstream), MMP-9 (ERK target), and CRP reductions are consistent with ERK amplitude normalisation. Interactions: MEK inhibitors (trametinib, cobimetinib — oncology) + spirulina: additive ERK suppression in cancer cells plausible; insufficient evidence; oncology patients must consult their team.