signal transduction pathway regulation by signaling mucins by paul cullen at suny-buffalo
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Signal Transduction Pathway Regulation by
Signaling Mucins
by Paul Cullenat SUNY-Buffalo
Signaling Pathways Globally Regulate Cellular Decision-Making
When mis-regulated -> diseases (cancer)
“MAPK” Pathways
Regulators of the TNF Signaling Pathway Genome Biology (2007) 8, R104
Signaling Pathways Are Web-Like Information Networks
Typical Questions
What are the players in a signaling pathway?
How is a signal transmitted through a pathway with high fidelity
(specificity)?
How are multiple signals integrated into a cohesive response
(integration)?
Nutrient-Dependent Foraging Behaviors
T.B. Reynolds and G.R. Fink (2001) V291 p878
Filamentous Growth Biofilm Formation
Required for Virulence In Fungal PathogensRequired for Virulence In Fungal Pathogens
The Filamentous Growth (FG) Pathway
Cell-Surface Proteins:
Rho GTPase/PAK:
MAPK Cascade:
Transcription Factor:
The FG Pathway Shares Components With Other MAPK Pathways
Glycosylated
TransmembraneDomain
N…(698)S Q V S D T SV S Y T T S S S S V S Q V S D T PV S Y T T S S S S V S Q V S D T PV S Y T T S S S S V S Q V S D T PV S Y T T S S S S V S Q V S D T PV S Y T T S S S S V S Q V S D T SV P S T S S S S S V S Q V S D T PV P S T S S S S S V S Q (818) …C
Msb2 is a Signaling Mucin that Functions in the FG Pathway
Rho GTPaseInteraction
Tandem Repeats
Cullen, P.J. et al. G&D (2004)
Studying Signaling Mucins in a GeneticallyTractable Model May be Informative
Cullen, P.J. (2007)
What are Signaling Mucins Sensing?How do they become activated?
An Activation Mechanism for
Signaling Mucins
An Activation Mechanism for
Signaling Mucins
Immunoblot anti-HA for Msb2-HA Immunofluorescence Msb2-GFP
The Extracellular Domain of Msb2 Is Secreted
Aspartyl Protease Yps1 Processes Msb2
Immunoblot anti-HA for Msb2-HA
Relative levels of Msb2-HA S/P by immunoblot
ß-galactosidase assays
Extracellular Domain Is Inhibitory
Cleavage-Dependent Activation Mechanism for Signaling Mucins
Vadaie et al. J. Cell Biology (2008)
The extracellular domains of signaling mucins may be inhibitory.
Processing may represent a general activation mechanism for signaling
mucins.
Secreted/cell-associated aspartyl proteases may similarly process
mammalian mucins(Liaudet-Coopman et al. 2006).
Signaling Mucins and MAPK Specificity
Comparative Analysis of Msb2 and Hkr1
Genes encoding the two mucins appear to be differentially regulated.
The extracellular domians of both mucins are secreted. Msb2 is preferentially shed under nutrient-limiting conditions,whereas Hkr1 is not.
Msb2 has a Sho1-dependent function in cell polarization that isinhibited by Hkr1.
Overexpression of Hkr1 dampens the FG pathway in some mutants.
Overexpression of Msb2 and Hkr1Induce Different Outputs
DNA Microarray Analysis
Signaling Mucins Might Contribute to Specificity
Differential Mucin Function in MAPK Pathways
Pitoniak, Birkaya et al. (2009)
Many Pathways Control Filamentous GrowthMany Pathways Control Filamentous Growth
Secretion Profiling as an Approach to Identify New MAPK Regulatory
Proteins
Immunoblot anti-HA Quantitation of Immunoblot
4,750 ordered deletion mutants5,710 genes on overexpression plasmids
510 genes that influence Msb2 secretion
>10,000 transformations and immunoblots of >10,000 samples (~700 retests)
Secondary Screens to Focus in on Interesting Genes
Genes that influence Msb2 protein secretion
Genes that influence MSB2 expression
Differentiate between these classes using an MSB2-lacZ reporter
Comparing Expression and Secretion Profiles
Many Pathways Control Filamentous GrowthMany Pathways Control Filamentous Growth
How are different signals integrated into a complex differentiation response?
An Integrated Network of An Integrated Network of Filamentous Growth RegulationFilamentous Growth Regulation
Abdullah and Cullen, 2009; Chavel et al. (IN PRESS)
Polarity and Signaling
How does a MAPK pathway influence polarity?
Polarity and Signaling
Shed Mucins and FungalPathogenesis
Is Flo11, another transmembrane mucin, shed from cells?
What is the function of mucin shedding in microorganisms?
Flo11 is Secreted from Cells and Confers Cellular Lubrication
Flo11 Is Shed from Mats
Part of a Secreted Layer that Hydrates,Protects, and Regulates Mat Behaviors
Conclusions1. The signaling mucin Msb2 functions in the
filamentous growth pathway. 1. -2. Processing and release of the inhibitory domain
of Msb2 is required for MAPK activation.
3. Hkr1 and Msb2 may preferentially function in the HOG and filamentous growth pathways, respectively.
4. Secretion profiling can identify new regulatory features of secreted mucins and effector MAPK pathways.
5. Shedding of Flo11 contributes to cellular lubrication and the formation of a secreted “mucus” layer.
-model to understand signaling mucins
-general activation mechanism for signaling mucins
-secreted mucins may impact microbial pathogenesis