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Research

Research

How Kinases are Regulated

WNK kinases are the long-anticipated kinases involved Transepithelial transport and cell volume control regulation regulated by chloride and osmotic

Inactive WNK1 is an Asymmetric Dimer

 

Protein kinases are a very fascinating family of proteins because they are involved in transducing sensory inputs into responses, and yet, they are soluble and amenable to study. My group has benefited from a collaboration with Melanie Cobb, who has cloned out several important protein kinases. We have been able to contribute significant insights into the action of these kinases through structural and biochemical analysis, making use of a deep understanding in our group of how proteins work. Most important, we have determined the chemical logic of the very famous MAP kinase module (discussed below). Very recently, we have discovered that WNK kinases are regulated by macromolecular crowding and chloride ion, and thus are the long-anticipated protein kinases involved in cell volume regulation. There are several interesting ongoing projects associated both with the WNK kinases and with MAP kinase modules. These projects build upon exciting intellectual insights, and thus offer good leads to, and opportunities for, new discoveries. 

 

Cavities

The mouth of WNK1 is full of cavities

Water in Dimer

The big cavity is full of water. What does it mean?

We are testing whether hydrostatic pressure induces autophosphorylation of WNKs, with preliminary data that this is the case.

 

 

WNK Kinases.

WNKs are associated with familial hypertension. They are proving to be the long-anticipated kinases that respond both to osmotic stress and chloride to control cell volume. But how do they work?

New projects concerning WNKs:

  • Crystallization of WNK kinases-- Find out how they are regulated by crystallizing WNKs from creatures living in different environments.
  • Determine if they are activated  by osmotic pressure.
  • Use shotgun mass spectrometry to find other pressure sensitive kinases
Pressure Devices Being Used
 

Goniostat

Microfuge

Nitrogen Gas


 

 

RASOPATHY mutants in MAP2Ks.

We have discovered that MAP kinase modules work through precisely ordered reactions, and that protein-protein binding steps are important kinetically.  This system is producing paradigms that explain how phosphorylation and multiple reaction steps produce sigmoid behaviors, and the mechanisms in play are much more deterministic than originally anticipated. Our model is the Excursion Model. We have published on the p38 MAP kinase pathway and need to do a study of the ERK2 pathway.

Cancer mutants have been identified that occur in MAP2Ks, the central component of MAPK modules. We are presently analyzing the origin of misregulation of these cancer mutants by mass spectrometry and kinetic modeling. This is a work in progress.

What can go wrong in the cancer mutants?

Answer: Everything!

 

Projects concerning Rasopathie

  • Determine structures of MEK1-based rasopathy mutants.
  • Form complexes of MEK1 with its cognate substrate and activator

These projects will tell whether the proteins induce conformational changes in each other.

  
Rasopathy mutant changes the order of phosphorylation in the activation of MEK1