Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Summary

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Abstract

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

Introduction

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. In the mouse, this process begins at embryonic day (E) 9.5 with the formation of the frontonasal prominence and pairs of maxillary and mandibular processes, each of which contains post-migratory cranial neural crest cells. The lateral and medial nasal processes arise from the frontonasal prominence with the appearance of the nasal pits and eventually fuse to form the nostrils. Further, the medial nasal processes and maxillary processes fuse to generate the upper lip. Concurrently, palatogenesis is initiated with the formation of distinct outgrowths – the secondary palatal shelves – from the oral side of the maxillary processes at E11.5. Over time, the palatal shelves grow downward on either side of the tongue, elevate to an opposing position above the tongue, and eventually fuse at the midline to form a continuous palate that separates the nasal and oral cavities by E16.5¹.

These morphological changes throughout craniofacial development are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. For example, cell membrane receptors, such as subfamilies of transforming growth factor (TGF)-β receptors, including bone morphogenetic protein receptors (BMPRs), and various receptor tyrosine kinase (RTK) families, are autophosphorylated upon ligand binding and activation in cranial neural crest cells^2,3,4. Additionally, the G protein-coupled transmembrane receptor Smoothened becomes phosphorylated in cranial neural crest cells and craniofacial ectoderm downstream of Sonic hedgehog (SHH) ligand binding to the Patched1 receptor, resulting in Smoothened accumulation at the ciliary membrane and SHH pathway activation⁵. Such ligand-receptor interactions can occur through autocrine, paracrine, and/or juxtacrine signaling in craniofacial contexts. For example, BMP6 is known to signal in an autocrine manner during chondrocyte differentiation⁶, whereas fibroblast growth factor (FGF) 8 is expressed in the pharyngeal arch ectoderm and binds to members of the FGF family of RTKs expressed in the pharyngeal arch mesenchyme in a paracrine fashion to initiate patterning and outgrowth of the pharyngeal arches^7,8,9,10. Furthermore, Notch signaling is activated in both chondrocytes and osteoblasts during craniofacial skeletal development through juxtacrine signaling when transmembrane Delta and/or Jagged ligands bind to transmembrane Notch receptors on neighboring cells, which are subsequently cleaved and phosphorylated¹¹. However, there are other ligand and receptor pairs important for craniofacial development that have the flexibility to function in both autocrine and paracrine signaling. As an example, during murine tooth morphogenesis, platelet-derived growth factor (PDGF)-AA ligand has been demonstrated to signal in an autocrine manner to activate the RTK PDGFRα in the enamel organ epithelium¹². In contrast, in murine facial processes during mid-gestation, transcripts encoding the ligands PDGF-AA and PDGF-CC are expressed in the craniofacial ectoderm, while the PDGFRα receptor is expressed in the underlying cranial neural crest-derived mesenchyme, resulting in paracrine signaling^{13,14,15,16,17}. Regardless of the signaling mechanism, these receptor phosphorylation events often result in the recruitment of adaptor proteins and/or signaling molecules, which frequently become phosphorylated themselves to initiate intracellular kinase cascades such as the mitogen-activated protein kinase (MAPK) pathway^18,19.

The terminal intracellular effectors of these cascades can then phosphorylate an array of substrates, such as transcription factors, RNA-binding, cytoskeletal and extracellular matrix proteins. Runx2²⁰, Hand1²¹, Dlx3/5^22,23,24, Gli1-3²⁵, and Sox9²⁶ are among the transcription factors phosphorylated in the context of craniofacial development. This post-translational modification (PTM) can directly affect susceptibility to alternative PTMs, dimerization, stability, cleavage, and/or DNA-binding affinity, among other activities^20,21,25,26. Additionally, the RNA-binding protein Srsf3 is phosphorylated in the context of craniofacial development, leading to its nuclear translocation²⁷. In general, phosphorylation of RNA-binding proteins has been shown to affect their subcellular localization, protein-protein interactions, RNA binding, and/or sequence specificity²⁸. Furthermore, phosphorylation of actomyosin can lead to cytoskeletal rearrangements throughout craniofacial development^29,30, and phosphorylation of extracellular matrix proteins, such as small integrin-binding ligand N-linked glycoproteins, contributes to biomineralization during skeletal development³¹. Through the above and numerous other examples, it is evident that there are wide implications for protein phosphorylation during craniofacial development. Adding an additional level of regulation, protein phosphorylation is further modulated by phosphatases, which counteract kinases by removing phosphate groups.

These phosphorylation events at both the receptor and effector molecule levels are critical for the propagation of signaling pathways and ultimately result in changes in gene expression in the nucleus, driving specific cell activities, such as migration, proliferation, survival, and differentiation, which result in proper formation of the mammalian face. Given the context specificity of protein interactions with kinases and phosphatases, the resulting changes in PTMs, and their effects on cell activity, it is critical that these parameters be studied in a physiologically-relevant setting to gain complete understanding of the contribution of phosphorylation events to craniofacial development. Here, examples of two contexts in which to study phosphorylation of proteins and, thus, activation of signaling pathways during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves – both primary³² and immortalized³³. At E11.5, the maxillary processes are in the process of fusing with the lateral and medial nasal processes¹, thereby representing a critical timepoint during mouse craniofacial development. Further, maxillary processes and cells derived from the palatal shelves were chosen here because the latter structures are derivatives of the former, thereby providing researchers the opportunity to interrogate protein phosphorylation in vivo and in vitro in related contexts. However, this protocol is also applicable to alternative facial processes and developmental timepoints.

A critical problem in studying phosphorylated proteins is that they are easily dephosphorylated during protein isolation by abundant environmental phosphatases. To overcome this barrier, adaptations and modifications to standard laboratory methods that allow for isolation of phosphorylated proteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphorylated proteins. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various signaling pathways active during craniofacial development.

Protocol

All the procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Anschutz Medical Campus and performed in compliance with institutional guidelines and regulations. Female 129S4 mice at 1.5-6 months of age and housed at a sub-thermoneutral temperature of 21-23 °C were used for embryo harvests. A schematic workflow of the protocol is represented in Figure 1. See the Table of Materials for details r…

Representative Results

When attempting to characterize the phosphorylation of proteins isolated from mouse facial processes and/or cultured palatal mesenchyme cells, the representative results will ideally reveal a distinct, reproducible band following western blotting with an anti-phosphoprotein antibody that runs at or near the height of the corresponding total protein band (Figure 3). However, if extensive phosphorylation of the protein occurs, there may be a slight upward shift of the phosphoprotein band compa…

Discussion

The protocol described here allows researchers to probe critical phosphorylation-dependent signaling events during craniofacial development in a robust and reproducible manner. There are several critical steps in this protocol that ensure proper collection of data and analysis of results. Whether isolating phosphoproteins from mouse facial processes and/or cultured palatal mesenchyme cells, it is imperative to move quickly and efficiently while keeping all reagents and materials on ice when indicated. The low temperature…

Materials

Equipment
Block for mini dry bath	Research Products International Corp	400783
ChemiDoc XRS+ imaging system with Image Lab software	Bio-Rad	1708265	chemiluminescence imager
CO2 incubator, air jacket	VWR	10810-902
Dissecting board, 11 x 13 in	Fisher Scientific	09 002 12
Electrophoresis cell, 4-gel, for mini precast gels with mini trans-blot module	Bio-Rad	1658030
Hybridization oven	Fisher Scientific	UVP95003001
Microcentrifuge 5415 D with F45-24-11 rotor (Eppendorf)	Sigma Aldrich	Z604062
Mini dry bath	Research Products International Corp	400780
Orbital shaker	VWR	89032-092
pH meter	VWR	89231-662
Power supply for SDS-PAGE	Bio-Rad	1645050
Rectangular ice pan, maxi 9 L	Fisher Scientific	07-210-093
Stemi 508 stereo microscope with stand K LAB, LED ring light	Zeiss	4350649020000000	dissecting microscope
Timer	VWR	62344-641
Tube revolver	Fisher Scientific	11 676 341
Vortex mixer	Fisher Scientific	02 215 414
Water bath	VWR	89501-472
Western blot box	Fisher Scientific	NC9358182
Materials
Cell culture dishes, 6 cm	Fisher Scientific	12-565-95
Cell culture plates, 12 well	Fisher Scientific	07-200-82
Cell lifters	Fisher Scientific	08-100-240
CO2	Airgas	CD USP50
Conical tubes, polypropylene, 50 mL	Fisher Scientific	05-539-13
Dumont #5 fine forceps	Fine Science Tools	11254-20
Embryo spoon	Fine Science Tools	10370-17
Microcentrifuge tubes, 0.5 mL	VWR	89000-010
Microcentrifuge tubes, 1.5 mL	VWR	20170-038
Pasteur pipet, 5.75"	Fisher Scientific	13-678-6A
Pasteur pipet, 9"	VWR	14672-380
Petri dishes, 10 cm	Fisher Scientific	08-757-100D
Petri dishes, 35 mm	Fisher Scientific	FB0875711YZ
Pouches, transparent, polyethylene lining	Fisher Scientific	01-812-25B
PVDF membrane	Fisher Scientific	IPVH00010
Semken forceps	Fine Science Tools	11008-13
Small latex bulb, 2 mL	VWR	82024-554
Surgical scissors	Fine Science Tools	14002-12
Syringe filter, 25 mm, 0.2 μm pore size	Fisher Scientific	09-740-108
Syringe with luer tip, 10 mL	VWR	BD309604
Transfer pipet	Fisher Scientific	13-711-22
Western blot cassette opening lever	Bio-Rad	4560000
Whatmann 3MM chr chromatography paper	Fisher Scientific	05-714-5
Reagents
4-15% Precast protein gels, 10-well, 30 µL	Bio-Rad	4561083
β-glycerophosphate disodium salt hydrate	Sigma Aldrich	G5422-25G	stock concentration 1 M
β-mercaptoethanol	Sigma Aldrich	M3148-100ML
Bovine serum albumin, fraction V, heat shock tested	Fisher Scientific	BP1600-100
Bromophenol blue	Fisher Scientific	AC403140050
Complete mini protease inhibitor cocktail	Sigma Aldrich	11836153001	stock concentration 25x
DC protein assay kit II	Bio-Rad	500-0112
DMEM, high glucose	Gibco	11965092
E7, mouse monoclonal beta tubulin primary antibody, concentrate 0.1 mL	Developmental Studies Hybridoma Bank	E7	1:1,000
ECL western blotting substrate	Fisher Scientific	PI32106	low picogram range
ECL western blotting substrate	Genesee Scientific	20-302B	low femtogram range
Electrophoresis buffer, 5 L	Bio-Rad	1610772	stock concentration 10x
Ethanol, 200 proof, 1 gallon	Decon Laboratories, Inc.	2705HC	EtOH
Ethylenediaminetetraacetic acid, Di Na salt dihydr. (crystalline powd./electrophor.)	Fisher Scientific	BP120-500	EDTA
Fetal bovine serum, characterized, US origin, 500 mL	HyClone	SH30071.03
Glycerol (certified ACS)	Fisher Scientific	G33-4
HRP-conjugated secondary antibody, goat anti-mouse IgG	Jackson ImmunoResearch Laboratories	115-035-146	1:20,000
HRP-conjugated secondary antibody, goat anti-rabbit IgG	Jackson ImmunoResearch Laboratories	111-035-003	1:20,000
Hydrochloric acid solution, 6N (certified)	Fisher Scientific	SA56-500	HCl
Igepal Ca – 630 non-ionic detergent	Fisher Scientific	ICN19859650	Nonidet P-40
Isopropanol (HPLC)	Fisher Scientific	A451-1
L-glutamine	Gibco	25030081	stock concentration 200 mM
Methanol	Fisher Scientific	A454-4
p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9102S	1:1,000; anti-Erk1/2
PDGF-BB recombinant ligand, rat	Fisher Scientific	520BB050
PDGF Receptor β primary antibody	Cell Signaling Technology	3169S	1:1,000
Penicillin-Streptomycin	Gibco	15140122	stock concentration 100 U/mL, 100 µg/mL
Phenylmethanesulfonyl fluoride, 99%	Fisher Scientific	AC215740100	PMSF; stock concentration 100 mM
Phospho-p44/42 MAPK (Erk1/2) primary antibody	Cell Signaling Technology	9101S	1:1,000, anti-phospho-Erk1/2
Phospho-PDGF Receptor α /PDGF Receptor β primary antibody	Cell Signaling Technology	3170S	1:1,000
Potassium chloride (white crystals)	Fisher Scientific	BP366-500	KCl
Potassium phosphate monobasic (white crystals)	Fisher Scientific	BP362-500	KH2PO4
SDS solution, 10%	Bio-Rad	161-0416
Sodium chloride (crystalline/biological,certified)	Fisher Scientific	S671-3	NaCl
Sodium fluoride (powder/certified ACS)	Fisher Scientific	S299-100	NaF; aliquot for one time use; stock concentration 1 M
Sodium orthovanadate, 99%	Fisher Scientific	AC205330500	Na3VO4; stock concentration 100 mM
Sodium phosphate dibasic anhydrous (granular or powder/certified ACS)	Fisher Scientific	S374-500	Na2HPO4
Tissue culture PBS	Fisher Scientific	21-031-CV
Transfer buffer, 5 L	Bio-Rad	1610771	stock concentration 10x
Tris base (white crystals or crystalline powder/molecular biology)	Fisher Scientific	BP152-1
Trypsin	BioWorld	21560033
Tween 20	Fisher Scientific	BP337-500
Western blot molecular weight marker	Bio-Rad	1610374
Software
ImageJ software	National Institutes of Health
Animals
Female 129S4 mice			gift of Dr. Philippe Soriano, Icahn School of Medicine at Mount Sinai