Two protein kinases, K1 and K2, function sequentially in an intracellular signaling pathway. If either kinase contains a mutation that permanently inactivates its function, no response is seen in cells when an extracellular signal is received. A different mutation in K1 makes it permanently active, so that in cells containing that mutation a response is observed even in the absence of an extracellular signal. You characterize a double-mutant cell that contains K2 with the inactivating mutation and K1 with the activating mutation. You observe that the response is seen even in the absence of an extracellular signal. In the normal signaling pathway, does K1 activate K2 or does K2 activate K1? Explain your answer.

The order must be K2→K1, since the permanently active K1 allele (K1a) is able to propagate the signal onward even when its upstream activator K2 is inactive (K2i). The reverse order would have resulted in a failure to signal (K1a→K2i), since the permanently active K1a kinase would be attempting to activate a dead K2i kinase.

Explanation:

You characterize a double mutant cell that contains K2 with type I mutation and K1 with type II mutation. You observe that the response is seen even when no extracellular signal is provided. In the normal pathway, i f K1 activat es K2, we expect t his combinat ion of two m utants to show no response with or without ext racell ular signal. This is because no matt er how active K1 i s, it would be unable to act ivate a mutant K2 that i s an activit y defi cient. If we reverse the order, K2 activating K1, the above observati on is valid. Therefore, in the normal signaling pathway, K2 activates K1.