00:47We can see some accessory proteins that have come along and have started to form the

signaling complex on the inside of the cell.

00:55This includes a protein known as GRB2.

00:57And GRB2 is binding to phosphoytyrosines that are on the cytoplasmic side of the cell, on the
EGF receptor.

01:07That autophosphorylation that I described before has happened here.

01:11And this GRB2 has an SH2 domain that recognizes those phosphoproteins and binds to them.

01:17GRB2 binds to another protein known as SOS, which for our purposes here is insignificant.

01:23But what is significant is the next protein that binds which is known as RAS.

01:27Now RAS is a protein that’s very intimately involved in controlling the cell’s decision to divide.

01:34RAS is a protein that is very much like the G-protein that I described before in the beta-
adrenergic receptor.

01:40It’s of similar size and it also binds to guanine nucleotides.

01:46The guanine nucleotide that it binds to here is GDP.

01:50And when it gets activated, which is what’s happening in this process, the GDP is replaced by
GTP and RAS is now active to go do its thing.

02:00And active RAS prepares a cell for division.

02:05Well there’s still more to this pathway; RAS in turn goes to another protein known as RAF
which is a kinase and activates it.

02:14RAF puts a phosphate onto another protein known as MEK, which is a kinase which puts a
phosphate onto another protein known as MAPK, which is of course a kinase that puts phosphates
onto other proteins as we can see here.

02:27Now, the order of this for our purposes isn’t significant.

02:30What’s important though is what’s happening in the overall process.

02:34This cascade of phosphorylations that’s happening is causing a bunch of different proteins to

become active, and it’s affecting at the very bottom, some proteins that are known as transcription
factors.
02:47These transcription factors become activated by the addition of the phosphate.

02:51And they move into the cell nucleus as you can see here to co-activate gene expression.

02:58The genes that they’re activating will stimulate the cell to divide.

03:02So epidermal growth factor has favored the process of cell division.

03:06Well before I go further I need to say an interesting word about RAS.

03:10RAS is actually a family of related proteins, it’s not just one, there are actually several.

03:16Each of these proteins is monomeric, that differs from what we saw with the G-proteins before;
they had the heterotrimers that we had associated with the β-adrenergic receptor.

03:26RAS proteins can bind guanine nucleotides as we saw.

03:30And this human RAS that’s shown here is bound to a GDP.

03:35RAS swaps GDP for GTP when it gets activated by that signaling complex that I showed
earlier.

03:43Now like the α subunit of the β-adrenergic receptor, RAS is also a bad enzyme.

03:50It very slowly cleaves GTP to GDP, meaning that RAS turns itself off over time.

03:59Well that’s good, because you don’t want a cell continually turned on and continually dividing
because that becomes known as a cancer.

04:07What we said before was important for a cell to be able to turn off a signal just like it was
important to turn on a signal.

04:13And that’s particularly important for RAS as we have seen.

04:16Now RAS as I said, is a bad GTPase.

04:19It converts GTP into GDP and turns itself off.

04:24What happens if RAS can’t turn itself off? That happens sometimes.

04:28And it happens too frequently unfortunately.

04:30They are what are called point mutations or single base changes within the RAS
coding sequences that if they change, they can affect the ability of RAS to cleave GTP.

04:42If they inhibit RAS’s ability to cleave GTP, RAS is always left in the on state.
04:48Well, you saw from the last slide what happens if it’s left in the on state continuously, the cell
will continue to divide uncontrollably and that can lead to cancer.

04:56And RAS is implicated in numerous human cancers.

05:00Now other things need to be turned off as well.

05:02The RTK that was the EGF receptor that I talked about specifically here also has to be turned
off.

05:08How does it get turned off? Well, like the β-adrenergic receptor, it gets internalized into the cell
in a process known as endocytosis, thus removing it from being part of the signaling processes.

05:20The phosphatases that get stimulated in the schemes that I’ve shown before are proteins that
remove phosphates from other proteins.

05:27And this whole cascade that I showed before, there was a whole series of phosphorylated
proteins.

05:33A phosphoprotein phosphatase acting on them inactivates the entire pathway with a single
action.

05:40That’s pretty cool.

05:42Now you might wonder, how is it that phosphoprotein phosphatase itself gets inactivated? And
it turns out that it gets inactivated by an interesting phosphorylation process.

05:52I wanted to take a minute and show you that.

05:54On the right part of the screen you can see the phosphoprotein phosphatase that’s in green,
that is bound to a protein called Gm; that’s not a G-protein.

06:03It’s just a muscle protein called Gm.

06:05In the form that you see on the top, it’s active.

06:08But a phosphorylation of the Gm, causes the Gm to release the phosphoprotein phosphatase.

06:16That leaves the phosphoprotein phosphatase less active.

06:20You also see floating out there, an inhibitor.

06:23The inhibitor by itself doesn’t bind to the phosphoprotein phosphatase and inhibit it.

06:28Rather what it does, is it waits for the inhibitor to get itself phosphorylated.
06:34When the inhibitor gets phosphorylated, phosphoprotein phosphatase binds to it and is then
completely inactivated.

06:41So that’s how it gets inactivated.

06:43The question then is, when does it get inactivated? And it gets inactivated by action of protein
kinase A.

06:52Now think about this reciprocal regulation.

06:55This reciprocal regulation is causing one set of enzymes to become active on binding of one
hormone, and another to become inactive, and then flip them with the other hormone.

07:04So in the case of epinephrine for example that I talked about, it was stimulating proteins that
were important in breaking down glycogen.

07:12And insulin was important for stimulating proteins that were making glycogen.

07:16We see that that reciprocal regulation extends all the way down to turning off proteins like
phosphoprotein phosphatase.

07:23Pretty cool process.