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