Spatial landmarks regulate a Cdc42-dependent MAPK pathway to control differentiation and the response to positional compromise

Basu, Sukanya; Vadaie, Nadia; Prabhakar, Aditi; Li, Boyang; Adhikari, Hema; Pitoniak, Andrew; Chow, Jacky; Chavel, Colin A.; Cullen, Paul J.

doi:10.1073/pnas.1522679113

Cited by 29 publications

(71 citation statements)

References 104 publications

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“…Rim101p (Lamb and Mitchell, 2003;Hu et al, 2007), SAGA (Venters et al, 2011) and 420 Rpd3p(L) (Hu et al, 2007;Venters et al, 2011). GIC2 expression is also regulated 421 during mating (Roberts et al, 2000) Here and in Basu et al (2016) we identify cross talk between the polarity pathway 427 and the fMAPK pathway that impacts bud emergence and filamentous growth ( Fig. 7).…”

Section: Discussion 395mentioning

confidence: 74%

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Regulation of Bud Emergence by a MAPK Pathway

Prabhakar¹,

Chow²,

Siegel³

et al. 2019

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All cells establish and maintain an axis of polarity that is critical for cell shape and progression through the cell cycle. A well-studied example of polarity establishment is bud emergence in yeast, where the Rho GTPase Cdc42p regulates symmetry breaking at bud sites and the establishment of polarity by interacting with effector proteins. The prevailing view of bud emergence does not account for regulation by extrinsic cues or signal transduction pathways. Here, we show that the MAPK pathway that controls filamentous growth (fMAPK pathway), which also requires Cdc42p and the effector p21 activated kinase (PAK) Ste20p, regulates bud emergence under nutrient-limiting conditions that favor filamentous/invasive growth. The fMAPK pathway regulated the expression of polarity targets that included the gene encoding a direct effector of Cdc42p, Gic2p. The fMAPK pathway also stimulated GTP-Cdc42p levels, which is a critical determinant of polarity establishment. The fMAPK pathway activity was spatially restricted to bud sites and highest at a period in the cell cycle that coincided with bud emergence. Time-lapse fluorescence microscopy showed that the fMAPK pathway stimulated the rate of bud emergence during filamentous growth. Unregulated activation of the fMAPK pathway induced growth at multiple sites that resulted from multiple rounds of symmetry breaking inside the growing bud. Collectively, our findings identify a new regulatory aspect of bud emergence that sensitizes this essential cellular process to external cues.

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Section: Discussion 395mentioning

confidence: 74%

“…Scanning electron microscopy (SEM) was based on established methods (Piccirillo and 708 Honigberg, 2011) and performed essentially as described (Basu et al, 2016). For some 709 experiments, cells were grown for 16 h in liquid media at 30˚C.…”

Section: Scanning Electron Microscopy 707mentioning

confidence: 99%

Regulation of Bud Emergence by a MAPK Pathway

Prabhakar¹,

Chow²,

Siegel³

et al. 2019

Preprint

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“…Bem4p-HA K323A K328A was also defective for invasive growth and expression of the FUS1-HIS3 reporter (Fig. S9), which in strains lacking an intact mating pathway ( ste4 Δ) provides a readout of fMAPK pathway activity ( 39, 52, 58, 94, 95 ). Bem4p-HA K328A was also tested but did not show the same defects in MAPK signaling and invasive growth as Bem4p-HA K323A K328A .…”

Section: Resultsmentioning

confidence: 99%

“…Thus, Bem4p might direct Cdc42p, in some manner, to a pathway-specific complex containing Kss1p. Finally, bud-site-selection proteins, culminating on the Ras-type GTPase Rsr1p ( 56, 57 ) also regulate the fMAPK pathway but no other Cdc42p-dependent MAPK pathways ( 58 ). Therefore, at least three Cdc42p-interacting proteins, Msb2p, Bem4p, and Rsr1p regulate the fMAPK pathway.…”

Section: Introductionmentioning

confidence: 99%

Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

Basu¹,

González²,

Li³

et al. 2019

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Impact Sentence: Rho GTPase adaptors play unique and sequential roles in the 27 regulation of MAPK pathways. 28 29 Cdc42p Adaptors Regulate a MAPK Pathway Basu et al. 2 HIGHLIGHTS 30• Comparing Cdc42p-dependent MAPK pathways showed that the fMAPK 31 pathway had slow activation kinetics compared to the mating and HOG 32 pathways. 33 34• A collection of cdc42 alleles was tested for MAPK pathway functions. 35 § Cdc42p E100A , previously characterized as being specifically defective for fMAPK 36 signaling, showed reduced interaction with the fMAPK pathway adaptor Bem4p. 37 § Corresponding residues in Bem4p were identified that were required for 38 interaction with Cdc42p and fMAPK signaling. 39 40 • The polarity adaptor Bem1p regulated the fMAPK pathway. 41 § Bem1p regulated the fMAPK pathway by recruiting Ste20p to the plasma 42 membrane, cycling between an open and closed conformation, and interacting 43 with the Cdc42p GEF, Cdc24p. 44 45 • Different domains of Bem1p had different roles in regulating effector pathways. 46 § Bem1p may function as a multi-functional adaptor in some pathways and an inert 47 48 49• Bem4p and Bem1p regulated the fMAPK pathway in an ordered sequence. 50 § The data support a model where Bem4p recruits Cdc24p to GDP-Cdc42p, and 51 Bem1p directs GTP-Cdc42p to Ste20p at the plasma membrane. 52 § The bud-site GTPase Rsr1p regulates Cdc24p in the fMAPK pathway but does 53 not initiate signaling. 54 55 Cdc42p Adaptors Regulate a MAPK Pathway Basu et al. 3 ABSTRACT 56 Rho GTPases regulate cell polarity and signal transduction pathways to control 57 morphogenetic responses in different settings. In yeast, the Rho GTPase Cdc42p 58 regulates cell polarity, and through the p21-activated kinase Ste20p, Cdc42p also 59 regulates mitogen-activated protein kinase (MAPK) pathways (mating, filamentous 60 growth or fMAPK, and HOG). Although much is known about how Cdc42p regulates 61 cell polarity and the mating pathway, how Cdc42p regulates the fMAPK pathway is not 62 clear. To address this question, Cdc42p-dependent MAPK pathways were compared in 63 the filamentous (∑1278b) strain background. Each MAPK pathway showed a unique 64 activation profile, with the fMAPK pathway exhibiting slow activation kinetics compared 65 to the mating and HOG pathways. A previously characterized version of Cdc42p, 66Cdc42p E100A , that is specifically defective for fMAPK pathway signaling, was defective 67 for interaction with Bem4p, the pathway-specific adaptor for the fMAPK pathway. 68Corresponding residues in Bem4p were identified that were required for interaction with 69 Cdc42p and fMAPK pathway signaling. The polarity adaptor Bem1p also regulated the 70 fMAPK pathway. In the fMAPK pathway, Bem1p recruited Ste20p to the plasma 71 membrane, cycled between an open and closed conformation, and interacted with the 72 GEF for Cdc42, Cdc24p. Bem1p also regulated effector pathways in different ways, 73 behaving as a multi-functional adaptor in some pathways and an inert scaffold in others. 74Genetic suppression ...

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“…Detection of phosphorylated MAP kinase (P~Kss1p) by immunoblot analysis was performed as described [60][61][62] . Mid-log phase cells were harvested and pellets were washed once with water and stored at -80°C.…”

Section: Immunoblot Analysismentioning

confidence: 99%

Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton

Prabhakar¹,

Vadaie²,

Krzystek³

et al. 2019

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Transmembrane mucin-type glycoproteins can regulate signal transduction pathways. In yeast, signaling mucins regulate mitogen-activated protein kinase (MAPK) pathways that induce cell differentiation to filamentous growth (fMAPK pathway) and the response to osmotic stress (HOG pathway). To explore regulatory aspects of signaling mucin function, protein microarrays were used to identify proteins that interact with the cytoplasmic domain of the mucin-like glycoprotein, Msb2p. Eighteen proteins were identified that comprised functional categories of metabolism, actin filament capping and depolymerization, aerobic and anaerobic growth, chromatin organization and bud growth, sporulation, ribosome biogenesis, protein modification by iron-sulfur clusters, RNA catabolism, and DNA replication and DNA repair. A subunit of actin capping protein, Cap2p, interacted with the cytoplasmic domain of Msb2p. Cells lacking Cap2p showed altered localization of Msb2p and increased shedding of Msb2p's N-terminal glycosylated domain. Consistent with its role in regulating the actin cytoskeleton, Cap2p, and another Msb2p-interacting protein, Aip1p, were required for the enhanced cell polarization during filamentous growth. Our study identifies proteins that connect a signalling mucin to diverse cellular processes and may provide insight into new aspects of mucin function. Msb2p Interacting Proteins Prabhakar et al. 3 Msb2p Interacting Proteins Prabhakar et al. 4 many fungal species 24, 25 . Cells undergoing filamentous growth grow as branched filaments of elongated and connected cells 26-29 . One of the pathways that regulates filamentous growth is a Cdc42p-dependent Mitogen Activated Protein Kinase (MAPK) pathway commonly referred to as the fMAPK pathway 30, 31 . Msb2p functions at the plasma membrane to regulate the fMAPK pathway 4, 5 . Msb2p is a single-pass transmembrane protein with a highly glycosylated N-terminal domain (1185 amino acids) connected to a cytoplasmic C-terminal signaling domain (97 amino acids) by a transmembrane domain. The cytoplasmic domain of Msb2p binds directly to Cdc42p 20 . Cdc42p associates with the p21-activated kinase (PAK) Ste20p 32, 33 to regulate the fMAPK cascade [Ste11p, Ste7p, and Kss1p 34 ] that culminates in the phosphorylation/activation of transcription factors [Ste12p and Tec1p 35 ], which induce the expression of filamentation target genes 36, 37 . The glycosylation of Msb2p is related to its signaling function 38 . Under nutrient-limiting conditions, Msb2p is underglycosylated, which results in its proteolytic processing through a quality-control pathway called the Unfolded Protein Response (UPR) in the lumen of ER. The UPR upregulates the expression of an aspartyl protease, Yps1p that processes Msb2p in its extracellular domain 39, 40 . Proteolytic processing of Msb2p is required for activation of of the fMAPK pathway 40 . Another signaling mucin in yeast is Hrk1p, which regulates the Ste11p branch of the HOG pathway 41 . The HOG pathway has two branches that converge on the MAPKK Pbs2p. Hrk...

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Spatial landmarks regulate a Cdc42-dependent MAPK pathway to control differentiation and the response to positional compromise

Cited by 29 publications

References 104 publications

Regulation of Bud Emergence by a MAPK Pathway

Regulation of Bud Emergence by a MAPK Pathway

Functions for Cdc42p BEM Adaptors in Regulating a Differentiation-Type MAP Kinase Pathway

Proteins That Interact with the Mucin-Type Glycoprotein Msb2p Include Regulators of the Actin Cytoskeleton

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