Protein kinase B (PKB/Akt) is a key enzyme in the signaling pathway that leads t

Question

Protein kinase B (PKB/Akt) is a key enzyme in the signaling pathway that leads to cell growth. PKB is activated by a phosphatidyl inositol dependent protein kinase (PDK1), which phosporylates PKB at threonine 308. Active PKB is also phosphorylated at serine 473.

You are working in a lab studying cell growth in response to insulin-like growth factor (IGF-1). Your advisor has been unsuccessful in identifying the kinase responsible for phosphorylation at serine 473. In a moment of brilliance, you constrcuct genes encoding two mutant forms of PKB: one that carries a point mutation in the kinase domain, PKB-K179M, rendering it enzymatically inactive, and one that carries a point mutation in the PDK-1 reognition domain, PKB-T308A. You transfect these two constructs, as well as wild-type PKB, into cells that do not express their own PKB. You then expose the three transfected cell populations to IGF-1 and analyze the phosphorylation states of PKB using antibodies.                                                                                                                                                                                                           

Anti-PKB recognizes all three forms of PKB regardless of phosphorylation state. Anti-P473 specifically recognizes phosphoserine at position 473, and anti-P308 specifically recognizes phosphothreonine at position 308.

Interpret the data. What is the identitiy of the enzyme that phosphorylates serine 473 on PKB? If you acquire more data, design the appropriate experiment.

Akt Akt construct Akt T308A K179M GF anti-Akt anti-P473 anti-P308 1 2 3 4 5 6 Figure 15-29 Expression levels of various forms of Akt and their degree of phosphorylation in the presence and absence of IGF1 (Problem 15-130) Anti-Akt recognizes all three forms of Akt regardless of their phosphorylation state; anti-P473 specifically recognizes the phosphorylated serine at position 473; anti-P308 specifically recognizes the phosphorylated threonine at position 308.

Explanation / Answer

The identity is that mutant K179M has a mutation in kinase domain and still it has threonine 308 recognition domain is working, the anti p308 recognizes it but anti p473 doesn’t show any level (non-significant and IGF-1 is present too). The other mutant T308A got problem for recognition at threionine 308 so anti p308 cannot recognize. But P473 has a less significant appearance for IGF-1 presence. It means there is a probability that the serine 473 residue is present in kinase domain of Akt. We can say that because anti p473 recognizes the normal Akt when IGF-1 is present. In case of IGF-1 absent for all AKt normal, mutant K179M and mutant T308A, the bands for antiP473 are quite similar can be ignored easily.

But it further needed to be confirmed and that can get by Fluorescence Resonance Energy Transfer (FRET) Microscopy analysis.

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