Advanced Molecular Genetics-Biology 566 Phosphorylation and dephosphorylation - Regulation in the downstream signaling cascade
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Phosphorylation is a switch in cellular functioning

There are over 120 different protein kinases in yeast and it is anticipated that there will be over a 1000 identified in the human genome.

Phosphorylation adds negative phosphate groups to the hydroxyl groups found on serine, threonine and more rarely, tyrosine.

About one-third of the cytoplasmic proteins contain a covalently bound phosophate.

Phosphorylation states can modulate the activity of proteins: for example, those used in glycogen metabolism.

Protein Kinase A

Protein Kinase A is a broad spectrum kinase that adds phosphates to serines and threonines that have an RRxS/Tx sequence pattern.

PKA dimers have tissue specific dimer regulatory subunits that serve as pseudosubstrates C2R2. There are four isoforms of R subunits that can form heterodimers and three isoforms of the C subunits.

Binding of cAMP to PKA is coopertive such that the regulatory subunits are released and the C2 catalytic subunits are active as kinases.

The activity of phosphatases reduce transcription back to a basal state.

Phosphorylation of receptors by PKA can also desensitize them resulting not only in lower activation of G-alpha-s but also an increased interaction with G-alpha-i which stimulates Ras and subsequently ERK.

Actions of cAMP not mediated by PKA

Examples:

Olfactory ion channels.

Guanine nucleotide exchange factor, Epac.

Protein Kinase C

The protein kinase C is a family of proteins that all require phosphoserine for activation.

Some are activated by phorbol esters.

Phosphorylation by PKC occurs only at serines and threonines with RxxS/TxRx motif patterns.

PKC is activated in four steps after synthesis:

The regulationof PKC is clearly linked to the activityof PLC to release DAG (diacylglycerol). There are a number of lipid sources and a protein which activate PKC.

"Within minutes of the addition of phorbol ester there occurs an extensive redistribution. PKCalpha and epsilon concentrates at the cell margin, PKC-alpha accumulates in the ER, PKCbeta2 asssociates with actin-rich microfilaments of the cytoskeleton, PKCgamma in golgi organelles, and PKCepsilon attaches to the nuclear membrane."

Substrates that interact with C-kinase (STICKs) bind target PKCs to specific subcellular sites.

Receptors for activated C-kinase (RACKs) and proteins interacting with C-kinase (PICKs) are not substrates but show specificity among the various isoforms.

Phosphorylation can inactivate some kinases and activate phosphatases, thus altering the normal transctiptional flow, for example in the TPA response element (TRE):

TPA induces production of the cytokine interleukin-1 which is essential for inductioof clonal expansion, through activation of PKC0.

"This metabolic response is vital for first line host defence and is also implicated in the tissue destruction associated with chronic inflammatory diseases."

PKC plays a role in recruiting components necessary for oxidation and the respiratory burst.

Dephosphorylation