The Significance of Kinases in Biological Catalysis and Phosphorylation

Abstract

Kinases are important enzymes in many biological reactions as they catalyze the transfer of a phosphoryl group from ATP to an acceptor. Protein kinase A is a type of kinase that is activated by cAMP (Berg, Tymockzo, Gatto & Stryer, 2015). Measuring protein kinase A activity is important for accuracy when set amounts are used in subsequent experiments. A fast-reliable method to measure protein kinase A activity is with an indirect enzyme linked immunosorbent assay (Lin, 2015). Protein kinase A activity increases with increasing concentration, by measurement proportional to colour intensity.

Colour change is produced by the binding of secondary antibody-HRP conjugate and TMB substrate (ImmuneChem Pharmaceuticals Inc., n.d.).

Introduction

Enzymes are proteins that speed up the rate of a biological reaction without being used up in the reaction (Martin & McFerran, 2017). Kinases are a type of enzyme that catalyze the transfer of a phosphoryl group from ATP to an acceptor. In humans, there are more than 500 homologous kinases, making kinases one of the largest protein families (Berg et al.

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, 2015). The measurement of protein kinase activity is essential in the understanding of biochemical processes (Wang, Yang & Zang, 2015). Therefore, the quantification of kinase activity is important in a variety of experiments.

Kinase tools can be used to discover new information and advance drug discovery (Wang et al., 2015). Enzyme assays that measure catalytic function are especially valuable for kinase drug discovery. The assay can be used to estimate affinity, identify inhibitors, evaluate selectivity and characterize molecular mechanisms. Enzyme assays can also be used to screen compounds and evaluate their specificity.

By accompanying other research approaches including cellular assays and binding assays, enzyme assays can also be valuable in aiding in the discovery of new information (Haubrich & Swinney, 2016).

The protein kinase test measures protein kinase activity, by using an indirect enzyme-linked immunosorbent assay (ELISA) to determine the activity per ng of various types of protein kinases. This is done by measurement of colour change from a substrate attached to a secondary antibody which colour intensity is proportional to kinase activity (ImmuneChem Pharmaceuticals Inc., n.d.). The quantification of protein kinase activity is important for accuracy in subsequent experiments.

Kinases, as part of a very large protein family, are important regulators of cell function. Protein Kinase A(PKA) is a cAMP-regulated enzyme, that plays many important roles in biological processes (Kotani, 2012).

Materials and Methods

Use an indirect enzyme-linked immunosorbent assay to measure protein kinase A activity. Start with a microtiter plate with CREBtide-BSA bound to the bottom. CREBtide-BSA is used on the plate instead of only CREBtide because it is hard to bind small molecules onto the plate (CREBtide is a synthetic substrate for PKA).

Addition of protein kinase A

1. added 90ul of ADB+ BSA buffer (100diln- 3mL ADB + 30ul BSA) to well H (Refer to Appendix Figure 3 for letter labeling of microtiter plate)
2. added 60ul of the ADB+BSA buffer to wells G to C
3. added 2 ul of PKA to well H and serial dilute to well C (will start with a concentration of 0.75ul in well H and end with 0.5ul in well C) and moved 30ul per well and changing tips every 3-4 wells, 30ul taken out of the solution after mixing from well C and discarded (end up with 60ul of solution per well)
4. added 60ul of BSA/ADB solution to wells B and A (Wells A and B do not contain any enzyme and are negative controls. Well A has no primary antibody but has the secondary antibody, whereas well B has both primary and secondary antibodies)
5. plate incubated at 37 degrees Celsius for 30 minutes

Addition of Primary Antibody

1. microtiter plate washed three times with PBST and tapped on a paper towel to dry
2. addition of primary antibody to all wells except for A (in A add antibody dilution buffer of the same amount of added primary antibody), added 70ul of 2500diln (2ul primary antibody and 5ml of 1x antibody dilution buffer).
3. sat microtiter plate at room temperature for 1 hour (place in a drawer to minimize possible contamination)

Addition of Secondary Antibody

1. washed the microtiter plate three times with PBST, and tapped on a paper towel to dry
2. added 60ul of secondary antibody 100 times dilution to all wells (5ml AbDB + 5ul of secondary antibody)
3. sat for forty minutes at room temperature in a drawer (the secondary antibody is light sensitive)
4. Addition of TMB to produce a colour change
5. washed the microtiter plate four times with PBST, to ensure all secondary antibody was washed out, and tapped on a paper towel to dry
6. added 60ul of TMB to all wells and let sit for 30 minutes at room temperature (colour changes from clear to turquoise)

Addition of stop solution

added 180ul of 0.5 M HCl to all wells (colour changes from turquoise to yellow)

Analysis of Results

using a plate reader for TMB colour measurement (OD at 450nm), calculated relative kinase activity of PKA: OD (sample)-OD (negative/ng of kinase)

Results

As seen in Figure 1, with an increasing concentration of PKA, there is increasing activity as shown by the colour intensity changes on the plate which are proportional to kinase activity. The negative control wells (row A) show minor, insignificant protein kinase A activity. The secondary antibody (1) goat anti-Rabbit HRP 1/5000 dilution has greater activity than the secondary antibody goat-anti-rabbit HRP (3) 1/1000 dilution. At 2000 nl/assay crude PKA both secondary antibodies showed very similar protein kinase activity.

Figure 1: Graph of PKA activity relating to data from Table 1

Table 1: Crude PKA test, with CREBtide coating (coated on March 6, 2019), crude PKA (from ImmuneChem lab), kinase reaction at 37C for 30 minutes in ADB containing 100ug/mL BSA, primary antibody Rabbit anti P-CREBtide (1/2500 newly prepared 360mL, RT, 60 mins) and secondary antibody, goat anti-Rabbit HRP (3) 1/1000 dilution and goat anti-Rabbit HRP (1) 1/5000.

Crude PKA (nl/assay) OD GaR-HRP (3) GaR-HRP (1) Blank 0.1260 0.1040 0.000 0.1800 0.2536 8.230 1.097 2.136 24.69 1.778 3.000 74.07 2.792 4.176 222.2 3.672 4.400 666.7 4.216 4.716 2000. 4.324 4.344 Discussion

The PKA enzyme assay used in this experiment is an antigen-captured immunosorbent assay (ImmuneChem Pharmaceuticals Inc., n.d.). The enzyme-linked immunosorbent assay (ELISA) is a quick, easy and a very sensitive technique (see Appendix Figure 4). This technique utilizes an enzyme-linked antibody binding to a surface-attached antigen (Lin, 2015). ATP is added as a phosphoryl group, so that protein kinase A will phosphorylate the substrate (see Appendix Figure 2). The kinase core binds ATP and a target peptide (Lin, 2015).

As discussed by Sassone-Corsi (2012), "[r]Regulation of transcription by PKA is mainly achieved by direct phosphorylation of the transcription factors cAMP-response element-binding protein (CREB)" (Sassone-Corsi, 2012, para. 6). With the presence of ATP on the plate, the protein kinase A can interact and phosphorylate CREBtide (a PKA substrate). With anti-phosphosubstrate antibodies, the phosphorylated substrate is analyzed. The binding of secondary antibody-HRP conjugate causes a colour change due to the TMB substrate (OD at 450 nm). The color production is used to measure protein kinase A activity, as the intensity of colour is directly proportional to phosphorylation activity. In order to stop the colour production, stop solution, 0.5M HCl is added (ImmuneChem Pharmaceuticals Inc., n.d.).

PKA is also known as a cyclic AMP-dependent protein kinase, as it is activated by elevations in cAMP (Pittinger, Nestler, & Duman, 2012). The activation of PKA by cAMP is how most effects of cAMP in eukaryotic cells are achieved. PKA is a combination of two types of subunits, a regulatory (R) subunit, and a slightly smaller catalytic (C) subunit. Three isoforms of the C subunit and four of the R subunits are encoded in the genome of all mammals (Berg et al., 2015). With cAMP present, two molecules of cAMP bind to each of the regulatory subunits, causing the R2C2 complex to dissociate into an R2 subunit and two C subunits, leading to the activation of the catalytic subunits. Without cAMP, kinase activity is prevented as the catalytic subunits become masked by the regulatory subunits creating and enzymatically inactive R2C2 complex (Pittinger, Nestler, & Duman, 2012). PKA is a well-studied effector of cAMP signaling (Sassone-Corsi, 2012).

The study of PKA is important , as it regulates many biological activities. An increase or decrease from the PKA activity optimal level can alter cell fate specification and lead to abnormal  HYPERLINK " o "Learn more about Cell Proliferation" cell proliferation (Kotani, 2012). PKA is involved in the regulation of the developmental stage of cells. For small adjustments in cell regulation PKA also has isozymic forms that are used when necessary (Berg et al., 2015).

Protein kinases catalyze the phosphorylation of proteins at serine, threonine and tyrosine residues (Roskoski, 2007). This post-translation modification is important to most physiological processes. A valuable tool to identify new inhibitors are kinase enzyme assays. Inhibitors can be used as starting points for drug discovery and for learning more information about the physiological function of kinases. Targeting kinases could be used for drug discovery of many non-infectious diseases. Already, more than 30 medicines inhibit protein kinases, with their primary uses against cancer (Haubrich & Swinney, 2016).

Conclusion

The indirect ELISA is an easy, fast and reliable way to measure kinase activity. The production of colour from the substrate attached to the secondary antibody is proportionate to the protein kinase A activity. As the protein kinase concentration increases on the plate, its activity also increases (see Figure 1). The plates showed high activity with an increase in PKA as expected, therefore the CREBtide is evenly coated on the plate. Results are due to the protein kinase A, as negative wells appeared to have no kinase activity, and the increase in protein kinase activity is proportional to the amount of protein kinase added to each well.

The protein kinase test is important in quantifying protein kinase A as it has roles in many biological processes. Protein kinases have an important role within the cell in signalling and enzyme and protein regulation (Lu & Hirsch, 2005). Therefore, protein kinase A is used in various experiments. Protein Kinase A is activated by cAMP and used in signaling systems in eukaryotic systems. For example, "in the yeast Saccharomyces cerevisiae, PKA regulates cell growth and morphology in response to nutrient and stress signals" (Lu & Hirsch, 2005 p.1794). PKA also plays an important role in the fight or flight response, helping animals cope in stressful situations (Berg et al., 2015). "Most effects of cAMP in eukaryotic cells are achieved through the activation of PKA by cAMP" (Berg et al., 2015 p. 297).

Measurement of protein kinase A activity is important for accuracy in future experiments. Recommendations for further study include, increasing PKA concentration's, beyond what was tested in this experiment, to determine if protein kinase A activity continues to increase, decrease or levels off. As well as, testing to determine the optimal PKA concentration of various cellular functions.

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