Structural Biochemistry/Proteins/Protein Phosphorylation by Protein Kinases
Phosphorylation of protein side chains in posttranslational modification of proteins is incredibly evolved and is a method of creating diversity in the proteomes of eukaryotes. Protein phosphorylation also occurs in prokaryotes but is less pervasive in bacterial protein metabolism and regulation. Phosphorylation of proteins involves the addition of phosphate groups to a target protein via protein kinases to activate or inactivate a certain function in the body. Marking proteins by the addition of phosphate groups assigns the proteins a code, which can instruct the cell to perform a number of functions, such as to divide or grow. Side chains of proteins that are phosphorylated (addition of a PO4 group) most commonly are serine, threonine, and tyrosine, which reflects the nucleophilic behavior of their -OH side chains. Also capable of phosphorylation is the imidazole ring nitrogen of histidine side chains in proteins. The process of phosphorylation turns protein enzymes on/ off, which can either cause or prevent certain diseses, such as cancer or diabetes. Nearly one-third of all the potential 30,000 proteins in the human proteome are estimated to be substrates for phosphorylation at a particular stage in their life cycle of eukaryotic cells. Kinases are those ATP dependent phosphorylation enzyme catalysts, and protein kinases are the subset working on protein substrates. There are over 500 estimated kinases in the human proteome, which is termed the human kinome. It is known that about 20-30% of the proteins in human body are phosphorylated. However, the problem is sorting out which of the 500 different kinases is responsible to the specific phosphorylation activity.
Protein Kinase A (PKA)Edit
Protein kinase A is an enzyme that covalently attaches phosphate groups to proteins. It is also known as the cyclic AMP-dependent protein kinase. An extremely significant characteristic of protein kinase A is its ability to be regulated by the fluctuation of cyclic AMP levels within cells. Essentially, protein kinase A is responsible for all cellular responses due to the cyclic AMP second messenger. Cyclic AMP activates protein kinase A, which phosphorylates specific ion channel proteins in the postsynaptic membrane, causing them to open or close. Due to the amplifying effect of the signal transduction pathway, the binding of a neurotransmitter molecule to a metabotropic receptor can open or close many channels.
- === Protein Kinase B (PKB) ===
Protein Kinase C (PKC)Edit
Protein kinase C catalyzes the process of signals mediated by phospholipid hydrolysis. It is activated by the lipid second messenger, diacylglycerol. This lipid second messenger serves as the key initiation for most protein kinase C's. Protein kinase C isozymes consist of a single polypeptide chain that possesses an amino-terminal regulatory region and a carboxy terminal kinase region. The isozymes are categorized into various groups: conventional protein kinase Cs which are regulated by diacylglycerol, phosphatidylserine, and Ca^2+ in addition to novel protein kinase Cs which are regulated by diacylglycerol and phosphatidylserine. Activation of GPCR's, TKR's, and non-receptor tyrosine kinases can lead to protein kinase activation by stimulation of either phospholipase Cs to yield diacylglycerol, or phospholipase D to yield phosphatidic acid and diacylglycerol. Additionally, conventional protein kinase Cs are regulated by Ca^2+.
Regulation of enzymatic activity: p300/CBPEdit
Phosphorylation is used in various enzymatic activities. For example, proteins p300 and CBP (CREB binding protein), when phosphorylated, increase the activity of acetyltransferase. Acetyltransferase then stimulates histone acetylation that promotes the transactivation of genes controlled by p300 and CBP activity. Conversely, phosphorylation can also be triggered by acetylation.
Protein Kinase Inhibitors in celling transduction and in clinical useEdit
Protein Tyrosine Kinase InhibitionEdit
The mutation of protein tyrosine kinases (PTKs) can change the communication between cells to be more or less frequent and can cause the spread of diseases. These diseases include various forms of diabetes and cancers. These mutations either enhance or detriment the rate of phosphorylation within the different proteins.
The inhibition of PTKs will help prevent the spread of these diseases and is believed to not cause much harm to the normal cells. PTKs are inhibited through the usage of tyrphostins (TYRosine PHOSphorylation INhibitors) as they bind to the ATP or substrate of the PTKs. These inhibitors were not supported at first as they were believed to block functions that were needed for the cell. However, after extensive tests and trials, it was found that there were natural occurring inhibitors and were as selective as needed. Both ATP competitive and substrate competitive molecules are used to block the signals.
The first inhibitors used were natural occurring; these included quercetin, genistein, erbstatitin, and lavendustin. However, these natural PTK inhibitors were not very effective as they were not very selective and also inhibited Ser/Thr kinaeses. These served as the model for the design of more potent and selective PTKs. These were developed through the usage of ATP mimics and substrate mimics to test the competitiveness of the designed inhibitor. One PTK that was developed was STI-571, which is effective in treating certain tumors.
Gefitinib and Erlotinib are two PTKs that were developed to treat non-small cell lung cancel (NSCLC).
Death - Associated Protein KinasesEdit
Death-associated protein kinases (DAPk) are kinases that regulate cell death and can also be used to induced cell death. DAPk has the ability to act as a tumor suppressor because it can sensitize cells to many signals that a cell encounters as it undergoes tumorigenesis. Its ability to suppress tumors shows that it plays a key role in tumor development. The study of how it’s expressed can function as a diagnostic tool to help scientists better evaluate disease in its severity, progression and other factors. However, excessive levels or increased activity of DAP kinases can be harmful and can contribute to diseases associated with the brain.
One of the structural components of DAPk is a death domain, located on the C terminus of the protein kinase, followed by a tail of 17 amino acids rich in serine residues. These serine residues are a key feature of death domain-containing proteins. Deletion of this tail was determined to produce a mutant that showed more killing potential than if the tail were not deleted however, the C terminus tail negatively regulates the cellular functions of DAPk. These functions demonstrate that the death domain competes with the full length kinase.
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