28.4: The next step - Downsteam intracellular signaling

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Learning Goals

• Understand the Role of Scaffolding Proteins in Signal Localization:

  • Explain how scaffolding proteins such as AKAPs (A-kinase anchoring proteins) localize activated PKA and PKC to specific subcellular domains.
  • Describe how scaffolds can integrate multiple signaling enzymes—including kinases, phosphatases, and phosphodiesterases—to ensure signal specificity and prevent off-target phosphorylation.

• Review Activation Mechanisms of PKA and PKC:

  • Summarize the activation process of PKA via cAMP binding that releases the catalytic subunits from the inactive R₄C₄ holoenzyme.
  • Outline the activation of PKC at the membrane by DAG, Ca²⁺, and subsequent phosphorylation events, and explain how these processes prepare PKC for downstream substrate phosphorylation.

• Relate Receptor Tyrosine Kinase (RTK) Activation to Downstream Signaling:

  • Describe how ligand-induced dimerization and autophosphorylation of RTKs generate docking sites for downstream adaptor proteins (e.g., GRB2) via their SH2 domains.
  • Discuss how these interactions lead to the recruitment of guanine nucleotide exchange factors (e.g., SOS), which activate small G proteins (Ras) and initiate further signaling cascades.

• Explore Key Signaling Pathways and Kinase Cascades:

  • Identify how the products of activated membrane enzymes (cAMP, DAG, and IP₃) serve as second messengers to activate kinases like PKA, PKC, and subsequently other cascades.
  • Summarize the basic architecture and amplification of the MAPK cascade, including the roles of MAPKKK, MAPKK, and MAPK in transmitting and amplifying signals.

• Examine Feedback Regulation and Computational Modeling of Kinase Pathways:

  • Explain the concept of feedback inhibition within kinase cascades, using the MAPK cascade as an example where the final product (MAPK_PP) can inhibit the initial kinase (MKKK) to produce oscillatory dynamics.
  • Appreciate how computational models (e.g., using VCell) can simulate these cascades under different conditions, revealing dynamic behaviors like oscillations and ultrasensitivity.

• Understand Structural Features and Allosteric Regulation in Kinases:

  • Discuss common structural motifs (e.g., activation loops, catalytic loops) found in kinases and how phosphorylation induces conformational changes that activate enzyme function.
  • Compare structural features of kinases such as PKA, PKC, and AKT, noting how domains like the pleckstrin homology (PH) domain in AKT mediate membrane recruitment and regulation.

• Integrate Downstream Signaling Effects with Cellular Outcomes:

  • Explain how the activation of these signaling pathways ultimately leads to changes in gene expression, metabolism, cell proliferation, and other cellular responses.
  • Evaluate the clinical significance of dysregulated signaling—for example, how aberrant RTK activity and overexpression (e.g., HER2 in breast cancer) can drive disease and serve as targets for therapeutic intervention.

These learning goals are designed to help you build a comprehensive understanding of the downstream events in cell signaling, from the initial receptor activation through complex kinase cascades, and to appreciate how scaffolding and feedback mechanisms fine-tune these processes for specific cellular outcomes.

Intracellular signaling from activated PKA and PKC

We discussed the basics of protein kinase A holoenzyme (R4C4) activation, which occurs when cAMP binds to the regulatory subunit, thereby freeing the catalytic subunit C for activity. Likewise, we discussed the activation of PKC at the cell membrane by DAG, Ca²⁺ ions, and phosphorylation of key Ser/Thr in the protein. Where in the cell are the downstream protein targets of activated PKA and PKC? This is a much simpler question for RTKs.  Downstream signaling proteins bind to them through the interaction of their SH2 domains with the autophosphorylated receptor tyrosine kinases (RTKs). For activated PKA and PKC, it turns out that their location is controlled by scaffolding proteins, which bind to them either before or after their activation.

Let's discuss a particularly important scaffolding protein, the A-kinase-anchoring protein (AKAP). There are 13 classes containing 50 different members. These proteins bind PKA through its regulatory subunit, where cAMP can mediate the activation of the holoenzyme (R₄C₄). In addition, AKAPs can bind other signaling proteins, including PKC and phosphatases, the latter of which, in turn, counter-regulate signaling by phosphoproteins. For example, the bound phosphatases can dephosphorylate PKC, thereby deactivating it, as well as other downstream phosphoproteins. AKAPs can also bind phosphodiesterase, the enzyme that converts cAMP to AMP, returning signaling to baseline levels. AKAPs localize key signaling enzymes to sites where biologically appropriate protein targets are localized. Additionally, they prevent the indiscriminate phosphorylation of other off-target proteins throughout the cell. They may also allosterically regulate the activity of bound signaling proteins.

There are at least 50 A-kinase anchoring proteins or A-kinase anchor proteins (AKAPs) that bind the regulatory subunit of protein kinase A (PKA) and localize PKA to specific sites in the cell. By binding multiple signaling enzymes at specific sites, they integrate signaling pathways mediated by cAMP (for example) with others mediated by PKC (again, for example).

Here are some examples of AKAPs in humans (from UniProt). Note that one (12) also binds PKC

• 1, mitochondrial: Binds to type I and II regulatory subunits of protein kinase A and anchors them to the cytoplasmic face of the mitochondrial outer membrane;
• 6: Binds to type II regulatory subunits of protein kinase A and anchors/targets them to the nuclear membrane or sarcoplasmic reticulum;
• 7 isoforms, alpha and beta: Targets the cAMP-dependent protein kinase (PKA) to the plasma membrane, and permits functional coupling to the L-type calcium channel;
• 7 isoform gamma: targets cAMP-dependent protein kinase (PKA) to the cellular membrane or cytoskeletal structures;
• 8: Acts as an anchor for a PKA-signaling complex onto mitotic chromosomes, which is required for the maintenance of chromosomes in a condensed form throughout mitosis;
• 8-like: Required for cell cycle G2/M transition and histone deacetylation during mitosis and recruitment of signaling enzymes into the nucleus;
• 9: assembles several protein kinases and phosphatases on the centrosome and Golgi apparatus;
• 12: Anchoring protein that mediates the subcellular compartmentation of protein kinase A (PKA) and protein kinase C (PKC)
• 17A: Splice factor regulating alternative splice site selection for certain mRNA precursors. Mediates the regulation of pre-mRNA splicing in a PKA-dependent manner.

Figure \(\PageIndex{1}\) illustrates the localization/binding of signaling enzymes (PKA, PKA substrates, PDE, other kinases) to AKAPs.

Figure \(\PageIndex{1}\): Localization/binding of signaling enzyme (PKA, PKA substrates, PDE, other kinases). Calejo et al. Frontiers in Pharmacology 6:192 (2015) DOI: 10.3389/fphar.2015.00192. License CC BY 4.0

Note that some AKAPs can also bind to PKA substrates, facilitating their phosphorylation and minimizing the phosphorylation of incorrect targets.

AKAPs utilize an amphiphilic helix to interact with the R2 regulatory subunit of protein kinase A (PKA). Some AKAPs bind to just one of the regulatory subunits. Note that some AKAPs can also bind to PKA substrates, facilitating their phosphorylation and minimizing the phosphorylation of incorrect targets.

Figure \(\PageIndex{2}\) shows specific AKAP complexes in the heart that could be targeted for drug therapies.

Figure \(\PageIndex{2}\): AKAP complexes in the heart. Calejo et al. Ibid

Panel (A): Disruption of the AKAP18γ/δ-PLB (another phospholipase involved in signaling) interaction prevents PLB phosphorylation on Ser16 and dislocation from SERCA2 (Sarcoplasmic/endoplasmic reticulum calcium ATPase 2). This inhibits SERCA2 activation and consequently Ca²⁺ uptake into the sarcoplasmic reticulum

Panel (B): Disruption of the nesprin-1α /mAKAP interaction promotes AKAP/PKA complex dissociation from the perinuclear membrane and might be a strategy to reduce hypertrophy. Nesprin 1 is a protein that forms a linking network between organelles and the actin cytoskeleton to maintain the subcellular spatial organization.

Panel (C): Disruption of the connexin 43-ezrin interaction could prevent PKA-mediated phosphorylation, increasing inter-cardiomyocyte conductivity, which could be cardioprotective following myocardial infarction damage. Connexin is a gap junction protein. Ezrin is involved in the connections of major cytoskeletal structures to the plasma membrane.

Adding to the complexity of PKA activation and signaling, there are different forms of the regulatory subunits of the holoenzyme PKA. These include RIalpha (RIA), RIbeta (RIB), RIIalpha (RIIA), and RIIbeta (RIIB). They have different affinities for cAMP, the catalytic subunits Cs, and different AKAPs.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of the amphiphilic anchoring peptide AKAP-IS for AKAP binding to the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA (2IZX)

Figure \(\PageIndex{3}\): Amphiphilic anchoring peptide AKAP-IS for AKAP binding to the docking and dimerization (D/D) domain of the RIIalpha regulatory subunit of PKA (2IZX) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...pWKiYgQeGA32e6

The brown represents the RII dimer D/D domains of the regulatory subunit. The anchoring peptide AKAP-IS is shown in gray. In both, the side chains involved in the peptide's binding to the regulatory subunit domains are depicted as sticks and color-coded according to the hydrophobicity of the side chains. Green indicates the most hydrophobic. Rotate the model carefully to differentiate the side chains and note that the hydrophobic face of the peptide interacts with hydrophobic side chains projecting into a groove made by the two RII dimer D/D domains. Polar side chains in AKAP help target the correct isoform of the R subunit.

In addition to binding to some AKAP scaffolds, PKC also binds to Receptors for Activated C-Kinases (RACKs), heat shock proteins (HSPs), importins, and annexins (AnxA1, A2, A5, and A6). The interactions of activated PKC with RACK1 and downstream events are shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Interactions of PKC with RACK1 and downstream signaling. Meng et al. PLoS ONE · (2014) DOI: 10.1371/journal.pone.0109111. Creative Commons Attribution License - https://creativecommons.org/licenses/by/4.0/

The insect protein BR-C (Broad Complex) contains a DNA-binding domain, comprising two zinc fingers, which facilitates gene transcription activation, and a BTB domain that enables binding to RACK1. On binding the PKC:RACK1 complex, BR-C is phosphorylated at Ser373 and Thr406, after which it is translocated into the nucleus, where it binds DNA and activates gene transcription.

The binding of PKC to RACK1 stabilizes PKC, allowing it to phosphorylate its targets. PKC binds to RACK through its C2 regulatory domains. Binding may be to specific forms of PKC, including unphosphorylated, inactive, and activated phosphorylated forms, as well as to specific isozymes of PKC. RACK1 may also recruit PKC to the ribosome, and it inhibits the activity of SRC kinases, which will be discussed later. PKC activity occurs in many cellular locations, including the cell membrane, nucleus, Golgi apparatus, mitochondria, and cytosol. RACKS also bind and recruit other signaling proteins, including PLCγ, Src, and integrins. In addition to interactions between PKC and RACK mediated by the C2 domain, PKC can also localize through its C1 domain.

Structure of RACK1

RACK1 (317 amino acids) has a very interesting structure. It is a member of a family of over 100 proteins that have tryptophan-aspartate (WD) repeats that are 44-60 amino acids long and end in WD or a variant. It is homologous to the beta subunit of the heterodimeric Gαβγ signaling protein. RACK 1 interacts with proteins through a 7-bladed propeller structure that allows the binding of proteins with SH2 domains, pleckstrin homology (PH) domains, and C2 domains (PKCs). Figure \(\PageIndex{5}\) shows the WD repeats in human RACK1. Note that the N-terminal end of the WD repeat is often glycine-histidine (GH).

Figure \(\PageIndex{5}\): Sequence of RACK1 depicting WD-repeats. R. Adams, D.R., Ron, D. & Kiely, P.A. RACK1, A multifaceted scaffolding protein: Structure and function. Cell Commun Signal 9, 22 (2011). https://doi.org/10.1186/1478-811X-9-22; https://creativecommons.org/licenses/by/2.0 )

Figure \(\PageIndex{6}\) a model for the interaction of RACK1 and PKC-βII.

Figure \(\PageIndex{6}\): Model for PKC-βII and RACK1 interaction. Adams et al. Cell Communication and Signaling 2011, 9:22 http://www.biosignaling.com/content/9/1/22. Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)

Panel (A) shows the resting state with no interaction between RACK1 and inactive PKCbII. Panel (B) illustrates how the activation of PKC-βII results in its interaction with RACK1. Substrate binding and phosphorylation follow. R is a receptor, and L is its ligand.

Figure \(\PageIndex{7}\) shows how RACK1 can translate into the nucleus after ligand (L) gated activation of GPCRs (R) through adenylyl cyclase production of cAMP and activation of Protein Kinase A.

Figure \(\PageIndex{7}\)Model for cAMP/PKA-mediated nuclear translocation of RACK1. Adams et al. Cell Communication and Signaling 2011, 9:22 http://www.biosignaling.com/content/9/1/22. Creative Commons Attribution License. (http://creativecommons.org/licenses/by/2.0)

Panel (A) shows the resting state of RACK, which forms homodimers and heterodimers with the homologous Gβ subunit of the Gαβγ complex. Panel (B) illustrates how the activation of PKA leads to the dissociation of RACK, which can then translate into the nucleus, resulting in increased transcription of brain-derived neurotrophic factor (BDNF).

Figure \(\PageIndex{8}\) shows an interactive iCn3D model of a the human Rack1 (4AOW) color coded as in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{7}\): human Rack1 (4AOW) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...7WDaKw2dxGK87A

Downstream signaling from activated receptor tyrosine kinases

To review once again, when receptor tyrosine kinases get activated by binding a primary messenger such as a growth factor, the receptors dimerize, activating their cytoplasmic kinase domains. The activated RTK then autophosphorylates itself. The phosphorylated intracellular domain serves as a docking site for other cellular adaptor proteins that contain an SH2 domain. The bound adaptor protein binds other signaling molecules within the cell, leading to downstream propagation of the signal. Figure \(\PageIndex{8}\) shows some RTKs and downstream signaling events. We have already discussed the activation of the lipid kinase phosphoinositide 3-kinase (PI3K), which in turn leads to the activation of Akt (also known as PKB). In the rest of this section, we will focus on the next step after the activation of RTKs. We focus on the epidermal growth factor receptor (EGFR, also known as ErbB1).

Figure \(\PageIndex{8}\): Some RTKs and downstream signaling events Dev et al. Frontiers in Pharmacology, 12 (2021). https://www.frontiersin.org/article/...ar.2021.772510. DOI=10.3389/fphar.2021.772510. Creative Commons Attribution License (CC BY).

1. Downstream signaling from the epidermal growth factor receptor.

Figure \(\PageIndex{9}\) shows events associated with the activation of EGFR.

Figure \(\PageIndex{9}\): Near downstream signaling molecules after activation of EGFR.  Mattox TE, Chen X, Maxuitenko YY, Keeton AB, Piazza GA. Exploiting RAS Nucleotide Cycling as a Strategy for Drugging RAS-Driven Cancers. Int J Mol Sci. 2019 Dec 24;21(1):141. doi: 10.3390/ijms21010141. PMID: 31878223; PMCID: PMC6982188.  Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Once bound and activated by binding of growth factor protein signals, the intracellular domains of the now dimeric EGFR autophosphorylate themselves on selected tyrosine side chains. This then recruits a protein called Growth factor receptor-bound protein 2 (GRB2), which has an SH2 (Src Homology 2) domain that binds phosphotyrosine motifs in proteins. GRB2 acts as an adaptor protein, possessing a SH2 domain and two SH3 (Src Homology 3) domains that bind to proline-rich domains on other signaling proteins, including the protein Son of Sevenless homolog (SOS). GRB2 does not have enzymatic activity.

The adaptor protein GRB2 binds through its SH3 domain to the protein SOS, which then interacts with the protein Ras. This last member in the recruited complex is named because it causes Rat Sarcomas. There are many variants of these proteins, but three are key in humans: HRas, KRas, and NRas. Ras is perhaps the key member of the family of intracellular small G proteins that bind GDP and GTP, and are activated upon the exchange of GTP for GDP. The proteins also have intrinsic GTPase activity, as is found in the G_α protein of the heterotrimeric Gαβγ protein.

So, what does SOS do? The SOS bound to RAS facilitates the exchange of GTP for GDP on Ras, maintaining it in an active state. SOS is a member of another fascinating class of small proteins that catalyze the exchange of GTP for GDP. The family of GTP/GDP exchange proteins is called Guanine Nucleotide Exchange Factors (GEFs). We will explore this in the next section.

The EGFR-GRB2-SOS-Ras complex in the figure above looks somewhat similar to the structure of a GPCR-heterotrimeric G protein GαGβGγ complex, where Gα is also a GTP/GDP exchange protein with intrinsic GTPase activity. When the ligand binds to the GPCR, a conformational change ensues, which facilitates the exchange of GTP for GDP on the G_α protein, activating intracellular signaling.

Once RAS is activated (bound to GTP), it binds and activates key kinases in the cell, including the lipid kinase PI3K and RAF, a kinase shown in Figure \(\PageIndex{8}\). Control of RAS activity is critical in signaling. It is one of the most commonly mutated proteins in cancer cells. Mutations that inhibit the intrinsic GTPase activity of the protein prevent it from being deactivated, leading to unregulated cell growth, proliferation, and differentiation — hallmarks of cancer cells.

The domain structures of GRB2 and SOS are shown in Figure \(\PageIndex{10}\). The proline-rich domain (motif) is not shown in the figure.

GRB2 domain structure (green SH3 domains)

SOS1 domain structure

Figure \(\PageIndex{10}\): Domain structure of GRB2 and SOS1 (proline rich domain not shown in SOS)

Note

New genes encoding proteins with new functionalities can be created by duplicating and adding gene segments for different domains in a preexisting gene. As we have seen with the SH2 and SH3 domains, signaling proteins often share common domains. Table \(\PageIndex{1}\) below, adapted from the excellent book "Cell Signaling" by Lim, Mayer, and Pawson, illustrates some common domains found in signaling proteins.

Domains in Signaling Molecules
Domain	Binding Target	Cellular Process	Example protein	Pdb file (examples) Find your own in the PDB
Bromo	Acetyl-Lys	Chromatin reg.	BRD4	2YYN
C1	diacylglycerol	Plasma membrane recruitment	Raf-1	3OMV
C2	Phospholipid (Ca-dependent)	Membrane targeting, vesicle trafficking	PRKCA	3IW4
CARD	Homotypic Interactions	apoptosis	CRADD	3CRD
Chromo	Methyl-Lys	Chromo reg, gene transcription	CBX1	3F2U
Death (DD)	Homotypic inter.	Apoptosis	Fas	3EZQ
DED	Homotypic inter.	Apoptosis	Caspase 8	1F9E
DEP	Memb, GPCRs	Sig trans, protein trafficking	Dsh human disheveled 2	2REY
GRIP	Arf/Art G prot	Golgi traffic	Golgin-97 (Golga5)	1R4A
PDZ	C-term peptide motifs	Diverse, scaffolding	PSD-95 Or discs large homolog 4	1L6O
PH	Phospholipids	Membrane recruitment	Akt	1O6L 3CQW
PTB	Phospho-Y	Y kinase signaling	Shc 1 SHC-transforming protein 1	1UEF 1irs
RGS	GTP binding pocket of Galpha	Sig trans	RGS4	1EZT
SH2	Phospho-Y	pY-signaling	Src	4U5W
SH3	Pro-rich sequence	Diverse, cytoskeleton	Src	2PTK
TIR	Homo/Heterotypic	Cytokine and immune	TLR4	3VQ2
TRAF	TNF signaling	Cell survival	TRAF-1	3ZJB
VHL	hydroxyPro	ubiquitinylation	VHL	1VCB

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the GRB2-SH2 domain in complex with a high affinity phosphopeptide KPFpYVNVEF (1BMB)

Figure \(\PageIndex{11}\): GRB2-SH2 domain in complex with a high-affinity phosphopeptide KPFpYVNVEF (1BMB) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...gBEpnsN6D8yrR6

Grb2 exists in both a monomeric state, which can bind SOS, and a dimeric state, which can't. You would expect the equilibrium between the monomer and dimeric form to be highly regulated. When a phospho-tyrosine ligand is bound to Grb2 through its SH2 domain, the dimer dissociates. This also occurs on phosphorylation of tyrosine 160 (Y160) on Grb2, a post-translational modification found in human prostate, colon, and breast cancers.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of the GRB2 N-terminal SH3 domain complexed with a ten-residue proline-rich peptide (1135 Ac-VPPPVPPRRR-NH₂) derived from SOS (1GBQ)

Figure \(\PageIndex{12}\): GRB2 N-terminal SH3 domain complexed with a ten-residue proline-rich peptide (derived from SOS (1GBQ). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...k2jNGaHexaUmT8

We will explore the structure of the small G protein Ras in conjunction with the guanine nucleotide exchange factor SOS in the next chapter section.

After activation of Ras through GTP exchange for GDP, the GTP-Ras protein binds to and activates the kinase Raf. We will continue our exploration of that later in this section.

2. Downstream signaling from the insulin receptor.

When insulin binds to the receptor tyrosine kinase (RTK), it phosphorylates itself, which then leads to the binding of other proteins to the activated receptor and their phosphorylation. These lead to increased intracellular signaling, ultimately resulting in changes in gene transcription. We'll focus on a specific adaptor protein, the Insulin Receptor Substrate 1 (IRS1), a "scaffolding protein" that leads to the movement of the glucose transport protein GLUT4 to the cell surface, allowing for glucose uptake. These activities are shown schematically in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\): Binding to insulin stimulates the association of the receptor with downstream mediators, including insulin receptor substrate-1 (IRS-1) and PI3K. The insulin receptor can activate PI3K either directly, by binding to the p85 regulatory subunit, which produces PIP3, or indirectly, which leads to phosphorylation and the activation of AKT. Afterward, AKT phosphorylates the Ser9 site of GSK-3β, inhibiting its activity. The PI3K/AKT/GSK-3β signaling pathway is involved in insulin signaling transduction, and GSK-3β is regulated and controlled by insulin in this signaling pathway. Arneth et al. Int. J. Mol. Sci. 2019, 20, 2467; doi:10.3390/ijms20102467. Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Human IRS1 has two domains: a PH domain for binding to membranes through phosphorylated IP2 derivatives and an IRS/PTB domain that binds phosphotyrosine residues on proteins. The PTB and SH2 domains are the most common domains for binding phosphotyrosine residues on proteins. PTB binds to the NPXY motif, where X is a phosphotyrosine (pTyr).

We show a more detailed view in Figure \(\PageIndex{14}\), in part, to review many of the signaling proteins we have seen before, including PI3K and PDK1.

Figure \(\PageIndex{14}\): A more detailed view of the insulin receptor-mediated translocation of GLUT4 to the membrane.

Likewise, another review of PIP2 derivatives is warranted. After phosphorylation by the activated insulin receptor protein tyrosine kinase, IRS-1 binds phosphatidylinositol 3-kinase (PI3K), which causes phosphorylation of the 3'OH on phosphatidyl inositol (PI) in the inner leaflet of the membrane to form PI(3)P. PI3K is a member of a family of kinases that phosphorylates PIP2. The metabolic pathway centered on PI3K is one of the most mutated in human cancers. PI(3)P, in turn, recruits other inactive kinases to the membrane, including phosphoinositide-dependent kinase 1 (PDK1) and Akt, also known as protein kinase B (PKB). Figure \(\PageIndex{15}\) shows phosphorylated phosphatidylinositol derivatives.

Figure \(\PageIndex{15}\): Phosphorylated phosphatidylinositol derivatives

On binding of PI(3)P, PDK1 becomes an active kinase, which phosphorylates and activates Akt. Akt kinases are major Ser/Thr protein kinase that phosphorylates proteins involved in a host of cell activities, including the regulation of glucose transport, cell proliferation, and death. In the insulin signaling pathway, active (phosphorylated) Akt leads to the movement of the GLUT4 protein from intracellular endosomal vesicles to the cell surface, which offers a quicker way to import glucose into the cell than if Akt activates the GLUT4 gene expression. PDK1 phosphorylation of Akt2-T309 is required for insulin-stimulated Glut4 translocation. If T309 is mutated to A309 or if PDK1 is inhibited, GLUT 4 is not translocated to the cell membrane.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (1IR3))

Figure \(\PageIndex{16}\): Activated insulin receptor tyrosine kinase in complex with peptide substrate and ATP analog (1IR3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...j11LhWKCUnreV9

The dimeric form of the kinase is shown. ANP is shown in spacefill. The peptide substrate containing the interior tyrosine (stick) for phosphorylation is shown in blue. Three key tyrosines in the activation loop that are autophosphorylated (Tyr1158, Tyr1162, and Tyr1163) are shown and labeled in the right-hand monomer.

As with a protein kinase, the activation loop of the insulin receptor kinase domain is phosphorylated, resulting in a conformational change that enables the binding of ATP and the target protein. The activation loop becomes phosphorylated on Tyr1158, Tyr1162, and Tyr1163, with Tyr1163 being the key residue.

Downstream signaling from Cytokine-activated Receptors- The JAK/STAT pathway.

We now explore two signaling pathways mediated by the adaptor proteins JAK and STAT. These are abbreviations for the Janus Kinase (JAK) and the Signal Transducer and Activator of Transcription (STAT). These play key roles in embryonic development, stem cell maintenance, hematopoiesis (formation of blood cells), and immune cell signaling. This pathway is stimulated by cytokines and interleukins, protein modulators released by immune cells, as well as growth factors.

In general, there are five groups of cytokines:

• tumor necrosis factor alpha (TNF-alpha)
• Interleukin-1 family members (IL-1_
• Transforming growth factor-beta (TGF-beta)
• those that signal through RTKs (such as macrophage colony-stimulating factor (M-CSF)
• Chemokines
• cytokines that signal through JAK/STAT

In contrast to RTKs, which have kinase domains activated on receptor dimerization, cytokine receptors that work through JAK/STAT do NOT have kinase domains. On cytokine binding to its target cytokine receptor, the prebound, inactive Janus kinase becomes activated through conformational changes. The kinase domains autophosphorylate themselves in a trans fashion. The active kinase then phosphorylates the cytoplasmic tails of the cytokine receptors. This triggers further signal transduction reactions mediated by the binding of target signaling proteins to the cytoplasmic region of the phosphorylated cytokine receptor. To reiterate, the cytokine receptor is NOT a RTK with latent kinase activity. Instead, it becomes phosphorylated by the bound and cytokine-activated JAK. A portion of the pathway is illustrated in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): An overview of the JAK/STAT signaling pathway (A) and the domain structures of JAK (B) and STAT (C). Thomas et al. TBr J Cancer 113, 365–371 (2015). https://doi.org/10.1038/bjc.2015.233. Creative Commons Attribution-Non-Commercial-Share Alike 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/

Panel (A1) shows that the kinase JAK is bound constitutively in an inactive state to the cytokine receptor cytoplasmic region, not through its SH2 domain, but through its FERM domain (Panel B). The figure implies the cytokine receptor is dimeric in the absence of a ligand. On cytokine binding, conformational changes and repositioning of the bound JAK activate its kinase domain, which phosphorylates the C-terminal tails of the cytokine receptor. STAT monomers, through their SH2 domains, bind to the phosphorylated cytokine receptor, where they get phosphorylated by the active JAK. The phospho-STAT monomers form a dimer, dissociate from the complex, and translocate to the nucleus where they act as transcription factors. The Janus kinase is named after Janus, the two-faced Roman god of beginnings, endings, and duality, since JAK has two nearly identical JH kinase domains. One has kinase activity, while the other inhibits it.

Panel (B) shows the domain structure of JAK. The FERM domain facilitates binding of JAK to the cytokine receptor. The JH2 pseudokinase domain regulates the kinase activity of the JH1 kinase domain. The Ps show the site on JAK that is necessary for activation.

Panel (C) shows the domain structure of STAT. The SH2 domain binds phosphorylated tyrosines. The carboxy terminus transactivation domain is required for full transcriptional activation. P marks the conserved tyrosine residue whose phosphorylation is essential for STAT activation.

The pseudokinase domain of JAK interacts with the kinase domain on the same chain and prevents its activity in the inactive monomer and dimer. Cytokine binding to the cytokine receptor induces a conformational change that promotes the interaction of the pseudokinase domain on one JAK monomer with the same domain on another, thereby facilitating dimerization and freeing the kinase domains for activity. Figure \(\PageIndex{18}\) shows the pseudokinase domain (orange) interactions in the active JAK dimer.

Figure \(\PageIndex{18}\): The pseudokinase domain (orange) interactions in the active JAK dimer.

The kinase domain of each dimer is shown in red, and the pseudokinase domain is shown in orange. The activation loop (cyan) and ADP (spacefill, CPK colors) are shown in the red kinase domains. The orange pseudokinase domain is shown with a bound adenosine (not ADP), depicted in spacefill and CPK colors. However, it lacks the DFG motif required for catalysis. The phenylalanine cluster (F635, 657, 574) is labeled. The structure is actually for a mutant that has the V657F mutation that promotes dimerization and JAK activity. Hence, the V657F mutation is oncogenic.

JAK activity can be inhibited by the protein suppressor of cytokine signaling (SOCS). The transcription factor STAT activates transcription of the protein.  The SOCS protein, in a feedback inhibition loop, binds to JAKs and also to IFNGR1, which inhibits JAK activity.

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of the structure of the inhibitory protein SOCS1 in complex with JAK1 kinase domain (6C7Y)

Figure \(\PageIndex{19}\): Structure of the inhibitory protein SOCS1 in complex with JAK1 kinase domain (6C7Y). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...tqtfo2SQ5TxF58

The N-lobe of the JAK1 kinase domain is shown in cyan and the C-lobe in magenta. ADP (sticks) and Mg²⁺ (green) are shown in the interface between the lobes. SOCS1 is depicted in gray, except for the kinase inhibitor region, which is represented by spheres using CPK colors. It binds in the substrate binding groove, preventing access to the substrate.

Activated JAK activity and signaling do not stop simply with the activation of STAT. In addition to stimulating signaling through phosphorylated dimers of STAT, cytokines also activate other signaling pathways through the same receptors. Examples include the PI3K pathway, described in an earlier section, and the MAPK pathways, which are further detailed later in this section. Both the PI3K and MAPK pathways are activated by the binding of cytokine IL6 to its cytokine receptor. The mechanism of PI3K activation is somewhat unclear. The MAPK pathway is activated by a phosphatase called SHP2 for Src homology region 2 domain‐containing phosphatase 1. This protein binds to pTyr759 on the IL-6 receptor. As the binding of the cytokine activates the prebound JAK, it also activates SHP2, ultimately leading to the activation of signaling through Ras (a small G protein), which in turn activates RAF, a kinase. Figure \(\PageIndex{20}\) illustrates the combined signaling pathways for a cytokine receptor, involving the activations of STAT, Ras, and PI3K.

Figure \(\PageIndex{20}\): Schematic representation of the JAK-STAT pathway. Lee et al. Immune Netw. 2017 Aug;17(4):214-227. https://doi.org/10.4110/in.2017.17.4.214. pISSN 1598-2629·eISSN 2092-6685. Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/)

The cytokine receptor induces activation of JAKs after cytokine stimulation, following the phosphorylation of STATs. Furthermore, phosphorylated STATs undergo dimerization and translocate to the nucleus, where they activate transcription of target genes. SOCS, suppressors of cytokine signaling; PI3K, phosphatidyl inositol-3 kinase; Akt, protein kinase B; FOXO, Forkhead box protein O; mTOR, mammalian Target Of Rapamycin.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA (1BF5)

Figure \(\PageIndex{21}\): Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA (1BF5). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...VEjHZ5t3baKC37

The dimer chains are shown in brown and gold, except for those colored according to their secondary structure (helix, red; sheet, yellow). The backbone of the nucleotides is shown in spacefill cyan and gray. Zoom in to see noncovalent interactions between the bound DNA and protein monomers. Two phosphotyrosines labeled pTR701 are also shown. The DNA-binding domain of the STAT dimer has an immunoglobulin fold and forms a "C-shaped clamp" around the DNA. The domains colored by secondary structure are SH2 domains, each recognizing and binding to the phosphorylated Tyr 701 (labeled pTR701), a very interesting use of the SH2 domain.

Figure \(\PageIndex{22}\) shows an interactive iCn3D model of the active Janus Kinase (JAK) dimer complexed with the intracellular domains (spacefill) of the interferon lambda(a cytokine) receptor (7T6F).

Figure \(\PageIndex{22}\): Active Janus Kinase (JAK) dimer complexed with the intracellular domain of the interferon lambda(a cytokine) receptor (7T6F). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9s3VBNMxsk7WM7

Domain/protein Coloring

• Red: PTKc-JAK-rpt2 kinase catalytic domain
• Orange: PTK-JAK1-rpt1 pseudokinase domain
• Yellow: FERM F2
• Magenta: FERM C-JAK1
• Cyan: SH2
• Brown: FERM F1
• Navy Blue and Blue spacefill: cytoplasmic domains of the cytokine receptor interferon lambda receptor 1 dimer
• The gold large sphere represents interferon bound to the extracellular domain of the interferon lambda receptor 1 dimer

Figure \(\PageIndex{23}\) shows a model combining the active Janus kinase (JAK) containing the intracellular domain of the interferon alpha receptor 1 dimer (navy and blue spacefill) with AlphaFold models of the extracellular and transmembrane domain of the interferon-alpha receptor (Q8IU57).

Figure \(\PageIndex{23}\): AlphaFold model of Interferon lambda receptor 1 Extracellular Domain and transmembrane domain (Q8IU57) with Active Janus Kinase (JAK) dimer complexed with cytokine receptor intracellular domain (7T6F)

Again, to reiterate as we did above, the cytokine receptor (in the figure above, the interferon lambda receptor) is NOT an RTK with latent kinase activity. Instead, it becomes phosphorylated by the bound and cytokine-activated JAK, which is resident in the cytoplasm.

Here is a link to a second iCn3D model, which shows the phenylalanine cluster that promotes pseudokinase domain interactions in the active JAK dimer.

Downstream Intracellular Signaling through Src Family Kinases - Membrane-associated non-receptor tyrosine protein kinases

Another family of intracellular protein kinases, the Src family, is often activated by the upstream activation of various receptors, including GPCRs, RTKs, cytokine receptors, as well as integrins and adhesion receptors, which we explore in a later chapter section. We have already touched on them when we discussed proteins containing the src homology domains SH2 and SH3. Src, an intracellular Tyr kinase (MW 60,000), is the founding member of this family of protein kinases. Src is activated when it binds to a phosphorylated membrane receptor through its own SH2 domain.

Src has many names, including Proto-oncogene tyrosine-protein kinase Src, proto-oncogene c-Src, pp60c-src, and p60-Sr. These membrane-associated, non-receptor tyrosine kinases regulate cell proliferation, differentiation, apoptosis, migration, metabolism, and cytoskeleton organization. They are one of the major classes of intracellular kinases that are activated after the upstream activation of the membrane receptors mentioned above, including GPCRs, RTKs, cytokine receptors, as well as integrins and adhesion receptors. They, in turn, activate further downstream protein kinases by phosphorylation. They even phosphorylate the upstream membrane protein, which leads to their activation.

There are 10 members in the Src family: Src, Frk, Lck, Lyn, Blk, Hck, Fyn, Yrk, Fgr, and Yes. They all share the same expanded domain structure shown in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\): The domain structure of Src (panel A) and its activation (panel B). Hsu et al, Cancers 12(6):1361. DOI: 10.3390/cancers12061361. CC BY 4.0

They have an SH4 N-terminal region that can be post-translationally modified with fatty acids (myristoylation and palmitoylation), which can anchor it to the membrane. The spacer SH2-SH3 spacer is intrinsically disordered and differs in sequence among members of the Src family. Two key phosphorylation sites (Tyr416 and Tyr527) are important. In inactive Src, Try 527 is phosphorylated, allowing it to bind to the SH2 domain. The SH2-kinase linker also binds to the SH3 domain. This occludes the active site region and prevents the phosphorylation of Try 416 in the "activation loop" of the SH1 kinase domain. When Try 527 is dephosphorylated, a conformational change occurs that opens the binding site, allowing autophosphorylation of Tyr 416 and subsequent activation. Hsu et al, Cancers 12(6):1361. DOI: 10.3390/cancers12061361. CC BY 4.0

Figure \(\PageIndex{25}\) shows an interactive iCn3D model of the Human tyrosine-protein kinase C-Src in complex with AMP-PNP (2SRC)

Figure \(\PageIndex{25}\): Human tyrosine-protein kinase C-Src in complex with AMP-PNP (2SRC). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...L45honKx6rrEu7

pY527 is shown in stick and labeled. It binds to the SH2 domain (151-248) shown in cyan. The SH3 domain is in magenta, and the kinase domain is shown in gray. The activation loop in the kinase domain is depicted in red, with the phosphorylated tyrosine residue (pY416) required for activation highlighted in stick representation and labeled. ANP is shown in spacefill.

We have now seen the structure of many kinase domains. Figure \(\PageIndex{26}\) shows an interactive iCn3D model of the alignment of human c-Src (452 amino acids) (2SRC) and Erk2 (362 amino acids), also called MAPK1 (2Y9Q), a protein kinase, which will be explored at the end of this section

Figure \(\PageIndex{26}\): Aligned structures of human c-Src (2SRC) and Erk2 (2Y9Q). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ETS1puViPtrQu7

Red shows conserved sequences, blue is aligned (but not conserved), and gray is non-aligned. Use the "a" key to toggle between the states.

After much discussion of the binding of p-Tyrosine side chains to the SH2 domain, we now present Figure \(\PageIndex{27}\), which shows an interactive iCn3D model of a phosphotyrosine peptide bound to the SH2 domain of Fer tyrosine kinase (6KC4)

Figure \(\PageIndex{27}\): Phosphotyrosine peptide bound to the SH2 domain of Fer tyrosine kinase (6KC4). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...3eqrcT7NwcHpr8

The SH2 domain is shown in gray. The phosphopeptide (DEpYENVD) is shown in cyan, with the labeled pY shown in stick representation. The side chains on the SH2 domains interacting with the pY are shown in stick and labeled.

We are about to explore the last but incredibly important downstream kinases activated in signal transduction cascades, the mitogen-activated protein kinase (MAPK) cascade. It could also be referred to as the Erk cascade. Before we do that, we will present parts of three pathways mediated by activated RTKs to refresh your minds, and also because the more you see key players in the pathway, the more you start to remember the names, structural features, and functions of the signaling molecules.

Figure \(\PageIndex{28}\) provides a brief overview of signaling through activated RTKs, which proceeds through the adaptor protein and the GEF SOS, ultimately leading to the activation of Ras, a key small G protein. Ras, in turn, activates a protein (MAPKKK) in the MAPK cascade.

Figure \(\PageIndex{28}\): Simplified cartoon showing the activation of the MAPK cascade protein MAPKKK. Cordover E, Minden A. Signaling pathways downstream to receptor tyrosine kinases: targets for cancer treatment. J Cancer Metastasis Treat 2020;6:45. http://dx.doi.org/10.20517/2394-4722.2020.101. Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/),

Figure \(\PageIndex{29}\) shows an abbreviated version of the activation of the lipid kinase PI3K and, through the activation of PDK1 and AKT, the activation of two major kinase complexes, mTORC1 and mTORC2, which we will explore in a separate chapter section.

Figure \(\PageIndex{29}\): Activation of mTORC1 and mTORC2, through activation of PI3K, PDK1 and AKT. Cordover E, Minden A. Signaling pathways downstream to receptor tyrosine kinases: targets for cancer treatment. J Cancer Metastasis Treat 2020;6:45. http://dx.doi.org/10.20517/2394-4722.2020.101. Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/)

Finally, Figure \(\PageIndex{30}\) shows the combined activation of both the MAPK (ERK) cascade pathway and the mTOR complex through GPCR signaling using the adaptor protein Grb2.

Figure \(\PageIndex{30}\): Combined activation of both the MAPK (ERK) cascade pathway and the mTOR complex through GPCR signaling using the adaptor protein Grb2.  Yang et al. Journal of Cellular and Molecular Medicine 22(6). DOI: 10.1111/jcmm.13447. License CC BY 4.0

Downstream Signaling through the Mitogen-Activated Protein Kinase Cascade

Active upstream kinases like PKA, PKC, and RTKs phosphorylate target proteins and, in doing so, change their activities. The usual protein targets are kinases, which become active on phosphorylation. They, in turn, activate other kinases, resulting in a complex cascade and amplification of the original signal. Often, the end product of such a cascade is a phosphorylated transcription factor that can alter gene expression. Perhaps the most described of these cascades is the Mitogen-Activated Protein Kinase (MAPK) pathway. Mitogens are chemical substances that stimulate mitosis (cell division). The MAPK system has three layers and a complex nomenclature (until you become accustomed to it). The end (downstream) product of the cascade is the enzyme mitogen-activated protein kinase (MAPK). It can be phosphorylated several times to produce MAPKP or MAPKPP, where the last Ps in the abbreviation signify the number of added phosphate groups.

The kinase immediately upstream of MAPK, which phosphorylates it, is abbreviated MAPKK (for mitogen-activated protein kinase kinase) or MAP2K. MAPKK (MAP2K) is activated by yet another upstream kinase called MAPKKK or MAP3K. If these are also targets of another upstream unrelated kinase, they would be abbreviated MAP3KPP, for example.

Now, of course, there are families of the MAPK cascade enzyme, each with its own name. Figure \(\PageIndex{31}\) shows the names of four different mammalian MAP3Ks leading to the activation of 5 different MAP2Ks, which lead to the activation of 4 different MAPKs. Some of these enzymes are so widely discussed in textbooks and journal articles that it is good to remember them specifically with their alternative names. These include the MAP3K enzymes Raf (Rapidly Accelerated Fibrosarcoma) and MEK, and the MAPK enzymes ERK (Extracellular Related Kinase) and JNK (c-Jun N-terminal Kinase)

Figure \(\PageIndex{31}\): Simplified overview of the mammalian MAPK cascades https://commons.wikimedia.org/wiki/F...-mammalian.png. Creative Commons Attribution-Share Alike 3.0 Unported

Figure \(\PageIndex{32}\) shows another representation of the MAPK cascade with some different enzyme names and added phosphates shown in circles.

Figure \(\PageIndex{32}\): Another representation of the MAPK cascade. Journal of Cardiovascular Development and Disease 6(3):27. 2019. 10.3390/jcdd6030027. CC BY 4.0

One way to organize a seminar on a complex topic is to follow these three steps: tell your audience what you will cover, tell them the information, and then tell them what you have covered. Following that advice, we present in Figure \(\PageIndex{33}\) an integrated view of signaling, starting from the membrane and moving inward to three enzymes in the MAPK kinase cascade, RAF (a MAP3K), MEK (a MAP2K), and ERK (a MAPK). Upstream signaling to the MAPK cascade comes from RTKs, GPCRs, and Ca²⁺ signaling, which we will discuss later.

Figure \(\PageIndex{33}\): Upstream signaling to the MAPK cascade from RTKs, GPCRs, and Ca²⁺ signaling Albert-Gasco et al. International Journal of Molecular Sciences 21(12):4471 (2020). DOI: 10.3390/ijms21124471. License CC BY 4.0

The sequence of events is:

• binding of an external signal to a membrane receptor and activation of the receptor kinase
• phosphorylation of receptor kinase and interaction with an activator GTP-binding protein such as Ras
• binding of activated G-protein to and activation of a mitogen-activated protein kinase kinase kinase (MAPKKK)
• MKKK phosphorylates and activates another kinase, MAPKK
• MKK phosphorylates and activates mitogen-activated protein kinase, MAPK
• MAPK phosphorylates inactive transcription factors or other proteins, thereby activating them. Unfortunately (from a naming point of view), when the activated proteins are themselves protein kinases, they are called mitogen-activated protein kinase-activated protein kinases (MAPKAPK)

There are seven types of MAPKs, four conventional and three atypical. Four typical ones are described in the table below.

Activator GTP-binding protein	Ras:GTP
MAPKKK or MAPK3	Raf-1A/B c-Mos	MEKK1-4 DLK MLK2	MEKK1-4 DLK MLK2	MEKK2/3 Tpl-2
MAPKK or MAPK2	MEK1,2	MEK4,7	MEK3,6	MEK5
MAPK or MAK	ERK1,2	JNK1-3	p38	ERK5
MAPKAPK	RSK 1-4 MNK2 MSK 1,2	MK2,3	MSK1,2 MK2,3	RSK1-4
An eventual Protein Target	c-Jun	c-Jun

MAP Kinase System from Cell Signaling

Structurally, these proteins are similar in size and domain structure to those of other kinases. Figure \(\PageIndex{34}\) shows an interactive iCn3D model of the alignment of MEK 1 (4U7Z) and ERK2 (5NHJ)

Figure \(\PageIndex{34}\): . (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Vq6RR7BA6o1Qb7

We now present multiple visual images of the MAPK cascade activation. Figure \(\PageIndex{35}\) shows two.

Details: EGFR_signaling_pathway.png. public domain

Figure \(\PageIndex{x}\): https://commons.wikimedia.org/wiki/F...ay_diagram.svg. Creative Commons Attribution-Share Alike 4.0 International

Figure \(\PageIndex{35}\): Two visual representations of the activation of the MAPK cascade through to activation of gene transcription.

As with protein kinase A and protein kinase C, signaling efficiency and specificity occur with minimal phosphorylation of incorrect targets when multiple signaling partners are scaffolded. This is also true of the MAPK cascade trio of kinases. Figure \(\PageIndex{36}\) shows the role of scaffolds KSR and Ste5 in MAPK signaling.

Figure \(\PageIndex{36}\): The role of scaffolds KSR and Ste5 in MAPK signaling. (A) In quiescent cells, an inactive KSR/MEK complex exists in the cytosol. (B) Upon stimulation of the cell, KSR translocates to the cell membrane and forms an active complex with phosphorylated Raf, MEK, and ERK. Activated ERK detaches from the scaffold with three outcomes: (1) ERK dimerizes in the cytoplasm, where the dimer remains or translocates to the nucleus; (2) ERK translocates to the nucleus; (3) KSR acts as a platform where ERK dimers are assembled and the new complex can interact with substrates in the cytoplasm. (C) Schematic of the yeast mating pheromone response pathway. Witzel et al. Front. Physiol., 21 December 2012 | https://doi.org/10.3389/fphys.2012.00475. Creative Commons Attribution License,

Computational Analyses - MAPK Cascade

You might think that the interconnected reactions of the MAPK cascade are complicated. However, as presented in the figures above, it consists of only three enzymes: MAPKKK (MAPK3 or MK3), MAPKK (MAPK2 or MK2), and MAPK. We added complexity by converting the actual enzymes from an inactive state to an active state by phosphorylation. In reality, this pathway is simple compared to the complete signal transduction pathways it is part of, and even simpler compared to the whole catabolic and anabolic pathways that we will see in the next sections.

We have discussed kinetics in earlier chapters and shown how it can be used to gain a more comprehensive understanding of an enzyme and its regulation and control. We also need to extend such kinetic analyses to entire pathways. We can do that using the VCell. Let's examine two different models of the MAPK cascade using VCell. One interesting feature is the regulation of the pathway. We have observed in our study of metabolism that the end product of the pathway often regulates the pathway itself. This makes sense since if the end product is abundant, it would be biologically sensible for the product to inhibit the first enzyme in the pathway, thereby avoiding the production of more of the ultimate end product. Of course, that inhibition would be relieved as the concentration of the end product falls. Hence, there is a temporal sense to the inhibition.

Let's run two Vcell models for the MAPK Cascade, one with no end product inhibition and one with inhibition of the first step. Since we are dealing with linked kinases, the inhibition of the first enzyme (MAP3K=MKKK) and hence the first reaction (MKKK ↔ MKKK_P) is not mediated by a chemical product of the last enzyme (MAPK_PP) but by phosphorylation of the first enzyme (MKKK) by the last (MAPK_PP).

MAPK Cascade - Model 1: No feedback inhibition of the MAPK cascade by inhibition of the first step (MKKK ↔MKKK_P) by the "end product" of the cascade (MAPK__PP) try quick

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MODEL 

MAPK Cascade - no feedback inhibition

Initial Condition - See simulation results

Select Load [model name] below

Interactive Element

Select Plot to change Y axis min/max, then Reset and Play  |  Select Slider to change which constants are displayed.  For this model, select Vm, Km, Ki and I |  Select About  for software information.

Move the sliders to change the constants and see changes in the displayed graph in real-time. 

Time course model made using Virtual Cell (Vcell), The Center for Cell Analysis & Modeling, at UConn Health.  Funded by NIH/NIGMS (R24 GM137787); Web simulation software (miniSidewinder) from Bartholomew Jardine and Herbert M. Sauro, University of Washington.  Funded by NIH/NIGMS (RO1-GM123032-04)

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The various concentration vs. time curves in the output graph should make intuitive sense. There are no surprises!

Now let's add a twist. What if the last active enzyme in the pathway, MAPK that is doubly phosphorylated (MAPK_PP), the "final product" of the cascade, can, in a feedback reaction, inhibit the very first reaction of the cascade, MKKK → MKKK_P, through inhibiting phosphorylation. Run the simulation to find out!

MAPK Cascade - Model 2: Feedback inhibition of the MAPK cascade by inhibition of the first step (MKKK ↔MKKK_P) by the "end product" of the cascade (MAPK__PP)

---

MODEL 

MAPK Cascade - With feedback inhibition

 Initial Conditions:  See simulation results

Select Load [model name] below

Interactive Element

Select Plot to change Y axis min/max, then Reset and Play  |  Select Slider to change which constants are displayed.  For this model, select Vm, Km, Ki and I |  Select About  for software information.

Move the sliders to change the constants and see changes in the displayed graph in real-time. 

Time course model made using Virtual Cell (Vcell), The Center for Cell Analysis & Modeling, at UConn Health.  Funded by NIH/NIGMS (R24 GM137787); Web simulation software (miniSidewinder) from Bartholomew Jardine and Herbert M. Sauro, University of Washington.  Funded by NIH/NIGMS (RO1-GM123032-04)

---

 

The various concentration vs. time curves in the output graph should make intuitive sense. There are no surprises!

Kholodenko2000 - Ultrasensitivity and negative feedback bring oscillations in the MAPK cascade. https://www.ebi.ac.uk/biomodels/BIOMD0000000010. Based on Kholodenko BN. Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades. Eur J Biochem. 2000 Mar;267(6):1583-8. doi: 10.1046/j.1432-1327.2000.01197.x. PMID: 10712587.

There is utterly no way to have predicted, using intuition or simple mathematical analyses, the oscillatory changes in the concentration of all the enzymes and their phosphorylated variants. Of course, the oscillating concentrations depend on the chosen initial concentrations and on the rate constants.

When enzymes are linked sequentially in signal transduction pathways, their actions are amplified by the preceding enzyme. Suppose the first kinase (MK3) activates 1000 molecules of the second kinase (MK2), and each of these activates 1000 of the last kinase (MAPK). In that case, the net effect of activating the first kinase is a million-fold amplification of the last! This causes the three-kinase pathway to be ultrasensitive to small changes in the first enzyme. Add other activating and inhibiting agents, and the net activities of the pathway components become even more complicated.

Just the simple feedback inhibition by the last enzyme of the first enzyme in this cascade can bring about the oscillation shown in Vcell model 2. Depending on the concentrations and rate constants used in the model, the oscillations can last from minutes to hours. These oscillations can generate waves of phosphoproteins that propagate through the cell's cytoplasm.

Here is a simplified animation of the MAPK cascade, illustrating changes in MKKK (red dots) and MAPK_PP (blue dots) with no feedback inhibition (left, Model 1) and with feedback inhibition (right, Model 2) in the cascade. (Animations produced by Shraddha Nayak and Hui Lui.)

Model 1: No feedback inhibition of the MAPK cascade	Model 2: Feedback inhibition of the MAPK cascade

You might expect similar oscillatory behavior in proteins, such as cyclins and cyclin-dependent protein kinases, that control the movement of cells through the cell cycle. We will explore this further in a subsequent section.

Summary

This chapter examines the intricate intracellular signaling events that follow receptor activation, focusing on how second messengers and protein kinases translate extracellular cues into precise cellular responses. It details the downstream pathways activated by the cAMP and DAG second messengers, the roles of key kinases, and the specialized protein complexes that localize and regulate these signaling events.

Key Points

• Activation of PKA and PKC:
  The chapter begins by revisiting the activation mechanisms of two major AGC family kinases. Protein kinase A (PKA) is activated when cAMP binds to its regulatory subunits within the inactive R₄C₄ holoenzyme, releasing active catalytic subunits. In parallel, protein kinase C (PKC) is activated at the cell membrane by a combination of diacylglycerol (DAG), Ca²⁺ ions, and phosphorylation events that modify key serine/threonine residues, relieving autoinhibition.

• Role of Scaffolding Proteins (AKAPs and RACKs):
  A central theme is the importance of scaffolding proteins, such as A-kinase anchoring proteins (AKAPs) and receptors for activated C-kinases (RACKs), in dictating the spatial organization of these signaling enzymes. AKAPs bind to the regulatory subunits of PKA and can also interact with other signaling molecules (including PKC, phosphatases, and phosphodiesterases), thereby localizing and integrating signaling complexes to specific cellular sites. This compartmentalization not only enhances the specificity of substrate phosphorylation but also prevents aberrant activation of off-target proteins.

• Integration of GPCR and RTK Signaling:
  The chapter compares and contrasts the intracellular signaling initiated by G protein-coupled receptors (GPCRs) with that initiated by receptor tyrosine kinases (RTKs). While GPCRs indirectly activate enzymes like adenylyl cyclase (producing cAMP) and phospholipase C (generating DAG and IP₃), RTKs become active kinases upon ligand-induced dimerization and autophosphorylation. Activated RTKs recruit downstream signaling proteins via their phosphorylated tyrosine residues and SH2 domain interactions, leading to additional phosphorylation cascades.

• Downstream Kinase Cascades and Signal Amplification:
  The activation of PKA and PKC triggers further phosphorylation events, which can amplify the initial signal dramatically. These kinases regulate a variety of cellular processes by phosphorylating specific target proteins, often in a cascade that involves multiple kinases. For example, activated RTKs can initiate the MAPK cascade, a three-tiered pathway (MAPKKK → MAPKK → MAPK) that culminates in the phosphorylation of transcription factors, leading to changes in gene expression.

• Clinical Relevance and Therapeutic Applications:
  The chapter also highlights the clinical significance of these signaling pathways. Aberrant signaling, such as overactivation of RTKs (e.g., HER2 in breast cancer), can lead to diseases like cancer. Targeted therapies, including antibody-drug conjugates and kinase inhibitors, are designed to modulate these pathways with high specificity.

Conclusion

Overall, the chapter provides a detailed view of how extracellular signals are converted into intracellular actions through the coordinated activity of second messengers, protein kinases, and scaffolding proteins. By exploring the activation and regulation of key kinases like PKA, PKC, and RTKs, and by understanding how these enzymes are precisely localized within the cell via scaffolding proteins like AKAPs, students gain insight into the complex network of signaling events that control cellular behavior. This knowledge is foundational for appreciating both normal physiology and the molecular basis of diseases, and it serves as a basis for the development of targeted therapeutic strategies.