Help! I don\'t understand any of this... The phosphorylation of a protein can in
ID: 169845 • Letter: H
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Help! I don't understand any of this...
The phosphorylation of a protein can influence its ability to interact with other proteins. These protein-protein interactions play a fundamental role in signal transduction pathways, and these interactions can be identified using numerous techniques, including Förster resonance energy transfer (FRET) (see Figure 15-15). Protein kinase A (PKA) has many substrates, one of which is glycogen phosphorylase kinase (GPK), which has a multimeric ()4 structure containing two regulatory subunits ( and ), the catalytic subunit, and the calcium sensor subunit, calmodulin.
a. You are using FRET to investigate PKA interactions with GPK and have cloned cDNA fusion constructs for three of its four different subunits (, , and ), all containing a fluorescent tag that when expressed excites at 480 nm and emits fluorescence at 535 nm. You also have cDNA encoding the catalytic domain of PKA fused to a tag that when expressed excites at 440 nm and emits at 480 nm. In the assay, if the PKA fusion protein interacts with one or more of the tagged GPK substrates, the transfer of energy from the PKA tag will excite the tag on the substrate, causing it to emit fluorescence at 535 nm, which can be detected.
O C Figure 15.UN02 Molecular Cell Biology, eighth edition CO 2016 W.H. Freeman and CompanyExplanation / Answer
Glycogen phosphorylase kinase (GPK) is involved in the breakdown of glycogen into glucose through phosphorylation. Initially, the addition of epinephrine to the cells cause the release of calcium ions into the cell and the activation of c-AMP. These calcium ions bind to the calcium sensor subunit, calmodulin of the GPK causing partial activation of the protein. Similarly, the activated c-AMP causes activation of PKA. PKA has the ability to bind to only the regulatory subunits ( and ) of the GPK. The activated PKA causes phosphorylation of regulatory subunits ( and ). The cells contain either fluroscent tagged , , or subunits which have the ability to get excited only through interaction with other activated protein causing Förster resonance energy transfer (FRET). However, PKA interacts with only the and subunits and as only the subunit is tagged, the highest peak (2nd peak) represents PKA+ subunit. The remaining peaks represent fluroscent tagged PKA (control, 1st), PKA+ subunit (3rd) and PKA+ subunit (4th). The values of fluroscence are not of significance because of the abscence of flurosccent tagged subunit in these cells which is the only subunit to receive the Förster resonance energy transfer (FRET) from the fluroscence tagged PKA.
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