What Is Protein Phosphorylation?

Protein phosphorylation: refers to the process of transferring the phosphate group of ATP to amino acid residues (serine, threonine, tyrosine) of protein by protein kinase or binding GTP under the action of signal, which is a kind of biological Ordinary regulation plays an important role in the process of cell signal transduction. Protein phosphorylation is the most basic, common, and important mechanism for regulating and controlling protein vitality and function.

Protein phosphorylation

Protein phosphorylation: refers to protein

Protein phosphorylation is the most basic, common, and important mechanism for regulating and controlling protein vitality and function. Protein phosphorylation occurs mainly on two amino acids, one is serine (including threonine) and the other is tyrosine. These two types of acid phosphorylated enzymes are not the same and their functions are different, but there are also a few bifunctional enzymes that can act on these two types of amino acids at the same time, such as MEK (mitogen-activated proteinkinase kinase, MAPKK ). The main role of serine phosphorylation is to reconstitute proteins to activate protein activity, mainly referring to enzyme activity. In addition to tyrosine phosphorylation, in addition to allostering and activating the activity of the protein, the more important function is to provide a structural gene for the binding protein to promote its interaction with other proteins to form a multi-protein complex. The formation of protein complexes further promotes the phosphorylation of proteins. Time and time again, the signal generated by the initial protein phosphorylation turned on and on. If a signal is initially generated that stimulates cell growth, this signal is eventually transferred to the nucleus, causing DNA replication and cell division.
Therefore, tyrosine phosphorylation and the formation of multiprotein complexes constitute the basic mechanism of cell signal transduction. Almost all peptide cell growth factors activate cells and stimulate cell growth through this pathway. Therefore, enzymes that catalyze the tyrosine phosphorylation of proteins, tyrosine kinases, have become key molecules in signal transduction mechanisms and in controlling cell growth. Tyrosine kinase and protein tyrosine phosphorylation also play a decisive role in tumorigenesis and growth. The development of many anti-tumor drugs has focused on such molecules.
Post-translational modification of proteins is an important area of protein chemistry research. The more you know about the details of protein structure, the wider the classification of protein post-translational modifications will be. Protein modifications include functional groups such as sugars, lipids, nucleic acids, phosphoric acid, sulfuric acid, carboxyl groups, methyl groups, acetyl groups, and hydroxyl groups that are linked to proteins by covalent bonds. Proteins have been modified and given new functions in terms of binding, catalysis, regulation, and physical properties. Protein phosphorylation is an important part of post-translational modification of proteins, and plays an important role in the activity of enzymes and other important functional molecules, the second messenger transmission and the cascade of enzymes. Protein phosphorylation is performed under the action of a series of protein kinases. This section mainly introduces the isolation and analysis of protein phosphorylases and the analysis method of phosphorylated proteins.

Roles in signal transduction: (1) Signal proteins in cells are mainly divided into two categories: one is phosphorylated under the action of protein kinases and covalently binds the phosphate group provided by ATP; the other is in signal Combined with GTP, GDP is usually replaced by GTP.

(2) The common feature of these two intracellular signaling proteins is that they are activated by obtaining one or several phosphate groups when the signal is reached, and they can be removed when the signal is weakened, thereby losing activity. In a signal relay network, phosphorylation of a signal protein usually causes downstream proteins to phosphorylate in sequence, forming a phosphorylation cascade.

(3) The phosphorylation of proteins is mainly concentrated on the tyrosine, serine, and threonine residues in the peptide chain. These residues have free hydroxyl groups and are not charged. When phosphorylation occurs, the protein will Having a charge, so that the structure changes, and further changes in protein activity, which is also the significance of protein phosphorylation.

Purification and Activity Analysis of Protein Phosphokinase

Protein phosphokinases are enzymes that catalyze the transfer of phosphate groups from phosphate donors to acceptor proteins. Usually, the -position phosphate (or other nucleoside triphosphates) of ATP is the donor. The International Biochemical Federation classifies protein kinases into the following categories based on the specificity of the amino acid of the receptor:
Phosphotransferases using protein ethanol groups as receptors are called protein serine or threonine kinases;
Phosphotransferases using phenyl as a phosphate receptor are called protein tyrosine kinases;
Phosphotransferases with His, Arg or Lys as receptors are called protein His kinases;
Phosphotransferases with Cys residues as receptors are called protein Cys kinases;
Phosphotransferases that use acetyl as a receptor are called aspart or glutamine kinase.

The first two types of enzymes are the most common, and many protein filament / threonine or tyrosine kinases have been purified. Using molecular biology techniques, more than 100 protein kinase genes have been cloned. Some have derived their corresponding amino acid sequences by determining their nucleotide sequences. In many cases, the specificity of the amino acid receptor of the cloned enzyme gene product cannot be determined directly, and it is generally obtained by comparing the sequence analysis with a protein kinase of known specificity. All known serine / threonine and tyrosine protein kinases have a common catalytic domain (approximately 270 amino acids). By comparing the sequence homology of the catalytic domains, the protein tyrosine kinase family can be divided into several subdomains. family. The protein filament / threonine kinase family is larger than the tyrosine kinase family. In addition, there are many reports about the activity of His, Lys and Arg-specific phosphorylases, but these enzymes have not been purified and their molecular structure is unclear.
(1) Purification of protein phosphokinase

The purification of protein kinases is divided into two types: micro-purification and mass-purification. The former is usually applied to cells cultured under special conditions, and the latter is generally used to special tissues and organs. The advantage of micro-purification is that the cloned cell line has a uniform cell type and is in a synchronized cell cycle. The intracellular components can be specifically labeled by radioisotopes. In cell culture, special factors can be used to study certain intracellular pathways. The disadvantage is that there are few raw materials and the amount of purified protein is limited.

The physical properties of a new protein kinase are generally unknown, so there is no fixed procedure for the purification of unknown protein kinases. In order to purify protein kinases from limited raw materials, the usual strategies are:

Prior to efficient affinity purification, traditional chromatography is used to remove the most abundant impurities;
Remove inhibitors and inactivating agents in the early stage of purification;
The processing volume should be reduced as soon as possible and dialysis should be avoided;
Purify as quickly as possible and avoid freezing.
Traditional chromatographic techniques for removing large amounts of foreign proteins include cation and anion exchange, hydrophobic and gel exclusion chromatography. These techniques are described in detail in many books on protein purification, and the ideal protocol for enzyme purification should allow a smooth transition between steps without dialysis. For example, hydrophobic chromatography is performed after the ion exchange step; gel exclusion chromatography is placed at the end to separate the protein and salt molecules, but it is necessary to add 0.2 to 0.5 mol / L salt to the gel exclusion medium to prevent the enzyme from binding to the medium non-specifically. Or aggregate. The easiest way to check the binding and elution behavior of enzymes on ion exchange and hydrophobic columns is to mix a small amount of medium with a small amount of protein kinase at the appropriate pH based on the physicochemical properties of the enzyme, and then analyze the enzymes in the supernatant after changing the ionic strength Of activity. After completing the above purification steps, affinity purification can be performed (commonly used affinity ligands are nucleoside triphosphates, substrates or effector molecules). The homogeneous product can only be achieved if the protein phosphokinase is purified by a factor of 104 to 106 by efficient affinity purification.

In order to purify protein kinases from cultured cells or tissues, inert systems such as the FPLC system of Pharmacia are generally used. The advantage of this system is that it has great inherent inertness and has the characteristics of maintaining macromolecule biological activity. The system contains a single piece of hardware in a unit and can be operated in cold rooms and at room temperature. In addition, a fast gradient (40-45 minutes) format and a wide range of high-efficiency gel and column systems are available. This is because under these conditions most proteins do not bind to the resin, including phosphatases that can inactivate kinases. However, many kinases, despite their isoelectric point 5.5, can still bind MonoS under neutral conditions, which may be due to their sulfonic acid group and ATP or substrate molecular structure is somewhat similar. Large volumes of extracts can be injected directly by the pump and subjected to two consecutive ion exchange separations. The FPLC pre-installed gel exclusion column can complete separation in 30 minutes, which is much faster than traditional gels. However, due to the small particle size of the column packing, the loading volume does not exceed 200 l, and the sample volume does not exceed 5-10 mg. The purification volume is small, and some properties are difficult to identify, so multiple purifications are required. Pharmacia introduced a new medium (Sephacryl HR) that is 5 times faster than traditional ultrafine gels. These gels greatly reduce the time required for gel filtration chromatography. After affinity purification, the purity of the enzyme can be detected by SDS-PAGE and its specific activity can be quantitatively determined.

(2) Analysis of protein phosphokinase activity The analysis of protein kinase activity is divided into two steps:
The phosphate group in the end-labeled nucleoside triphosphate nuclear donor (usually ATP, sometimes with GTP) is transferred to a protein or peptide substrate;
The phosphorylated substrate is separated and quantitatively analyzed.
The former is usually performed in solution, and both the enzyme and substrate are in the liquid phase. In order to remove free labeled nucleotides, the latter usually requires trichloroacetic acid precipitation, SDS-PAGE separation or binding of labeling substrate to cellulose membrane.
Sometimes the enzyme or substrate can be immobilized on a solid support. For example, the protein mixture is transferred to a nitrocellulose membrane by SDS-PAGE, then blocked, and placed in a solution containing the enzyme and labeled ATP. Or further, design a protein kinase analysis system (in situ analysis of enzyme activity), and fix the protein kinase and its substrate on the nitrocellulose membrane. This method can be equivalent to the standard liquid phase analysis method. Sensitivity and linear range. The analysis principle is that the sample containing protein kinase activity is first fixed on a nitrocellulose membrane (drop or vacuum suction), and then the filter is immersed in a suitable protein substrate solution, and the substrate protein is bound to the remaining membrane binding sites. Then, add isotope-labeled ATP, wash the unreacted ATP on the membrane after a certain period of time, and stop the reaction. The input is quantified by autoradiography or liquid scintillation counting, and the enzyme and substrate bound to the membrane have certain mobility The quantitative value of the two reactions represents the degree of phosphorylation.
Take the method for determining the activity of casein kinase II as an example:
Reagent: Buffer A25mmol / L Tris, 1mmol / L EDTA, 100mmol / LNaCl, pH8.0
Buffer B25mmol / L Tris, 1mmol / LEDTA, 200mmol / L NaCl, 10% glycerol (V / V), 1mmol / L DTT, pH8.0
Casein kinase II substrate hydrolyzed or partially dephosphorylated casein 10 mg / ml was dissolved in buffer A.
Casein kinase II reaction mixture 25mmol / L Tris, pH8.5, 100mmol / L NaCl, 10mmol / LMgCl2, 1mmol / L DTT, 0.1mol / L [V-32P] ATP (100Ci / mmol)
Steps:

1. Cut a nitrocellulose membrane of appropriate size and cut into 25px grids;
2. Wet the membrane with buffer A and place it on a filter paper soaked with the same buffer to make the membrane flat;
3. Buffer B (containing 1 mg / ml bovine serum albumin) was used to dilute the sample to a suitable concentration. The sample added at each point on the membrane had a kinase activity of 0.5-50 pmol units (1 pmol unit represents 1 pmol 32P per minute transfer Amount of enzyme), when the specific activity of the enzyme is 1mol / min · mg-1, 0.5 to 50ng of pure enzyme should be added at each point;

4. Use a micro-sampler to add 1 to 5 l of sample to the center of the cell above the membrane. After the droplets disappear, face the membrane to Parafilm sealing film containing buffer A (including 1 to 10 mg / ml substrate) (attached to Glass plate), and exposed to 23 ° C for 30 minutes in a humid environment. Large films can be sealed in plastic bags;

5. Wash the membrane in 100 ml of buffer A and place it on a new Parafilm membrane (with the reaction mixture solution 25 l / cm2), and act at 23 ° C for 5 to 30 minutes;
6. Wash the membrane 4 times with 100ml of buffer A, and perform autoradiography after air drying. Or cut into cubes for quantitative liquid scintillation counting.

The most common problem in the application of this method is overload of nitrocellulose membrane (referring to total protein or enzyme activity unit). The linear range of nitrocellulose bound BSA can reach 50 g / cm2 (equivalent to 5 g per spot), and the binding capacity can reach 500 g / cm2. If the sample itself causes protein overload, the BSA concentration in the dilution should be reduced. At the same time, care should be taken not to add too much enzyme, because exceeding 50 pM units is beyond the linear range of the method, and may affect the detection of adjacent spots on the nitrocellulose membrane. When this method is used for the detection of other protein kinases, the measurement conditions should be appropriately changed. Because the dot blot method of protein kinase activity is equivalent to the standard liquid phase method in terms of sensitivity, detection range, linearity, etc., it can replace the latter for routine detection. It also analyzes the distribution of protein kinase activity after natural gel electrophoresis and transfer electrophoresis, which can be used to analyze the expression changes of enzymes in different tissues at different developmental stages, cDNA clone analysis and mutant analysis.