Ras and Cancer Back to where we began: Protein structure combining Bioinformatics, protein structure with Kinemage, and the Map kinase path page 776-780

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Ras and CancerBack to where we beganProtein
structurecombining Bioinformatics, protein
structure with Kinemage, and the Map kinase
pathpage 776-780
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Ras was the first human oncogene found

• Take DNA from Human bladder cancer cells
• Put DNA into mouse cells (these cells already had
  other helpful mutations)
• Mouse cells become transformed cancerous
• Human Ras was found to be the oncogene found in
  the human bladder cancer cells

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We have a ClustalW analysis showing that the
number 12 amino acid G (glycine) can be replaced
with C (cysteine) and this causes human lung
cancer.The normal protooncogene Ras becomes an
oncogene
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K-Ras can have a point mutation- a single change
in the sequence
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Oncogenes- Commonly Are from Genes That act in
Cell signaling Paths-- Like the Ras Map
Kinase path
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Our Clustal W analysis of the amino acid change
in K RasFrom our lab The number 12 amino acid
is different. The First listing is normal,
second from cancer patient
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Change from normal G to cancerous C-symbolized by
G12C. Note no other weird animal has a change
(only mutant human protein)
No change here in human
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Why is there a different amino acid at position
12?
DNA coding strand sequence (you do not report the
template strand sequence) from a person with
cancer due to K Ras mutationfrom our prior work

• The Sequence Manipulation Suite Show
  TranslationResults for 5312 residue sequence
  starting "GGCCGCGGCG".       1  A  A  A  A  E  A 
   A  A  A  A  A  V  A  A  A  K  V  A  A  A        
   1 GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGA
  AGGTGGCGGCGGC      21  R  P  V  L  P  A  P  A  I 
   S  D  W  E  R  A  R  R  R  H          61 TCGGCC
  AGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGC
  ACTG      41  R  R  R  R  G  Q  R  L  S  G  S  Q 
   V  R  E  R  G  L  L  K       121 AAGGCGGCGGCGGGG
  CCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAGGCCTGCTGAA    
    
• 61  M  T  E  Y  K  L  V  V  V  G  A  C  G  V
    G  K  S  A  L  T       181 AATGACTGAATATAAACTTG
  TGGTAGTTGGAGCTTGTGGCGTAGGCAAGAGTGCCTTGACThe
  normal individual with a protooncogene has GGT
  not the dangerous TGT

Met is 1st Amino Acid
12th amino acid is C not G
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Use the Genetic Codefrom our text, for mRNA and
an Amino acidFor Ras induced cancer, change G
(glycine or gly) for a C (cysteine or cys) by
changing GGU in mRNA to UGU only 1
changeFig. 21-8, p 660 in 6th Ed
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In the normal cancer free individual with normal
K Ras, the K Ras mRNA with Glycine would
have.(using the Genetic Code)
GGU or GGC (also GGA or GGG code for
glycine) For Cysteine (C) in the mutant
oncogene K Ras the cancer patient would have
this sequence in their mRNA UGU or UGC So, the
first nucleotide base of the codon on mRNA
changes from a G to a U. This is due to a prior
change in the DNA of the cancer patient (a point
mutation).
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Follow change all the way throughnote that the
coding strand is always reported (same as mRNA
except replace U with T)
DNA
Coding strand
---------G changes to T -G-T-------
Template strand
---------C changes to A -C-A-------
mRNA
---------G changes to U -G-U------
Protein
------------12 Glycine changes to Cysteine
---------
And K Ras protein cannot be shut off, causes
cancer
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A POINT MUTATION IN THE DNA FROM G TO T
Normal protooncogene
Oncogene
---GGT---
---TGT---
coding
DNA
---ACA---
---CCA---
template
---GGU---
---UGU---
mRNA
AMINO ACID
---GLYCINE---
--CYSTEINE--
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Lets look at Ras structure and see where this
number 12 amino acid is locatedand why it is so
important-Ras is on and will cause cell
division if the Ras has GTP bound to it (ras is a
G Protein)Ras is off when GTP is broken down to
GDP. Ras itself breaks down GTP (a GTPase
reaction) but Mutant Ras cannot be activated to
breakdown GTP.So, mutant Ras stays oncausing
cancer (unregulated cell division)Put another
way, the mutation inhibits the GTPase activity of
Ras
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In general, Ras a central Beta sheetand 5 alpha
helices located on both sides of the Beta sheet.
GTP binds in the end of the Beta sheet
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12 Glycine In P/G1 loop and helps bind GTP
1 Amino Acid here
Gly12 here
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• Use the kinemage Ras file on web site, along with
  Kinemage, to view Ras structure
• View1 the backbone of the amino acid chain is
  in white, the bound GTP analogue in pink, and the
  Mg in yellow
• View2 is from one edge of the twisted beta sheet
  of this alpha/beta protein where GTP binds.
  Glycine 12 (where the most common mutation
  occurs) and Glycine 13 are labeled in green and
  are found on what is called the G1 or P loop.
• These 2 Glycines are located in a critical part
  of one of the main GTP-binding loops (the G1 or P
  loop). They are the two major sites of mutations
  that convert this enzyme into an oncogene - when
  these Gly's mutate to Cys, the GTP cannot be
  broken down down (GTPase activity of ras is
  reduced) so Ras stays in the "on" state more of
  the time- causing cancer.
• To see details of interactions at the binding
  site, choose View3 and turn on "interact.", which
  shows sidechains in cyan and weak H bonds in
  purple.