NOS and PKC

Question 1

3 forms of NOS

Answer 1

1-Neuronal: Constitutive form found in neurones 2-inducible: Found in macrophages/ kupffer cells/fibroplasts/vascular smooth muscle/endothelium. Upregulated by pathological stimuli 3. Endothelial; constitutive form. Also present in cardiomyocytes/renal mesangial cells/ osteoblasks/ platelets

Question 2

Important structural characteristics

Answer 2

• Dimeric Flavalin protein with homology to CYP proteins
• Contain a ZN atom with pairs of CXXXXC motifs at the dimer interface
• Monomers of NOS can ind CaM but not BH4 or L-Arginine => haem dependent dimerisation is essential to function
• Electron transfer from reductase domains enables No3 to bind O2 and form a Fe2+ dioxy species which may then recieve a second electron from BH4 or the reductase domain
• NOS enzymes perform two separate oxidation steps, one to form Nω-hydroxy-l-arginine and a second to convert this intermediate to NO.18
• The BH3• radical (or radical cation) can be recycled to BH4 by the NOS itself (using an electron supplied by the flavins). Alternatively, there is evidence that reducing agents such as ascorbic acid can reduce the BH3• radical back to BH4 (Asc·, ascorbate radical).

Question 3

Role within the cell

Answer 3

• Acts immediately in the local environment
• Reversiably bound to cysteines (such as heme) resulting in S-nitrosylated haemoglobin (still controversial)
• NO activates guanyl cyclase to begin producing cGMP this is posited to

In neurones: fascilitate Na+ influx/ excitation

In smooth muscle: Cause relaxation

Question 4

Cellular signalling of cGMP

Answer 4

Note: ROCK inhibits MyTP

Question 5

Kinase mediate regulation of NOS

Answer 5

Ser-1177 = positive modulation via increasing electron flux through reductase domains. Phosphorylation is induced when cells are exposed to oestrogens, vascular endothelial growth factor (VEGF), insulin, bradykinin or fluid shear stress.

Thr-495 is a negative modulatory site and is typically phosphorylated in endothelial cells, phosphorylation is associated with decreased electron flux and enzymatic activity. Phosphorylated by PKC and dephosphorylated by protein phosphatase 1.

Question 6

types of PKC and strucute function relationship

Answer 6

Pseudo substrate domains: Fits in the kinase domain to prevent activity when in inactive state

C2 Domain: Binds Ca2+ which initiates translocation to the membrane and contact with anionic phospholipids i.e. PIP2 or phosphatidyl serine

C1a: following C2 binding to membrane partners, bind to DAG forcing the pseudo substrate domain from the kinase domain leading to activation

c1b binds other partners

ORDER IS VITAL, 2xCa2+ must bind first

Question 7

PKC interactions: autoinhibitory loops and signalling

Answer 7

1st level:

PMCA: has PKCg phosphoylation site in the C-terminus. Phosphorylation interfered with inhibitor binding of calmodulin

NCX: Na+/Ca+ exchange is upregulated

Second level:

PLC(B) are phosphorylated by PKCa resulting in an inhibitory effect interfering with PIP2 pathways

PLD: potentiates PLD resulting in a stimulatory effect

AC: activates some isoforms of AC resulting in cAMP regulation of Ca2+ signaling events

NHE: protein phosphatases are upregulated

Question 8

Important functional characteristics of NOS

Answer 8

In nNOS and eNOS, Ca2+ is required for calmodulin binding whereas in inducible nos Calmodulin can bind in the calcium free form

When sufficient L-ARG and BH4 are present, Intact NOS dimers couple their haem and O2 reduction to the synthesis of NO

Question 9

Activated PKC

Answer 9

• Phosphorylate serine/threonine residues which are within close membrane proximity
• Specificity is conferred by adjacent amino acids to the threonine/serine residue