srcasm modulates egf and src-kinase signaling in keratinocytes

Srcasm modulates EGF and Src-kinase signaling in keratinocytes.

Weijie Li, Christine Marshall, Lijuan Mei, Leonard Dzubow, Chrysalene Schmults,

Michael Dans, and John Seykora*

Department of Dermatology

University of Pennsylvania Medical School

Philadelphia, PA 19104

*Corresponding author:

John Seykora M.D., Ph.D. Assistant Professor, Department of Dermatology University of Pennsylvania Medical School 211a Clinical Research Building 415 Curie Blvd. Philadelphia, PA 19104 Email: [email protected] ph 215 898 0170 fax 215 573 2033

JBC Papers in Press. Published on December 3, 2004 as Manuscript M406546200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 9, 2018

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Summary:

The Src-activating and signaling molecule (Srcasm) is a recently described activator and

substrate of Src-family tyrosine kinases (SFKs). When phosphorylated at specific

tyrosines, Srcasm associates with Grb2 and p85, the regulatory subunit of PI-3 kinase;

however, little is known about the role of Srcasm in cellular signaling. Data presented

here demonstrate that EGF receptor ligands promote the tyrosine phosphorylation of

endogenous and adenovirally-transduced Srcasm in keratinocytes, and that increased

levels of Srcasm activate endogenous SFKs, with a preference for Fyn and Src. In

addition, Srcasm potentiates EGF-dependent signals transmitted by SFKs in keratinocytes.

Tyrosine phosphorylation of Srcasm is dependent on growth factors and the activity of

EGFR and SFKs. Increased Srcasm expression enhances p44/42 MAP kinase activity and

Elk-1-dependent transcriptional events. Elevated Srcasm levels inhibit keratinocyte

proliferation while promoting specific aspects of keratinocyte differentiation. Lastly,

Srcasm levels are decreased in human cutaneous neoplasia. Collectively, these data

demonstrate that Srcasm plays a role in linking EGFR- and SFK-dependent signaling to

differentiation in keratinocytes.

Abbreviations: Ad-adenovirus, FCS-fetal calf serum, EGF-epidermal growth factor,

EGFR-epidermal growth factor receptor, KGF- keratinocyte growth factor (aka FGF-7),

MAP kinase-mitogen activated protein kinase, PHKs-primary human keratinocytes, PI-3

kinase-phosphoinositol-3-kinase, SCC-squamous cell carcinoma, SCIS-squamous cell

carcinoma in-situ, SFK-Src family kinase, Srcasm-Src activating and signaling molecule,

TGF-α- transforming growth factor alpha.


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Introduction:

Understanding the molecular mechanisms that regulate cellular proliferation and

differentiation are fundamental biological questions important within metazoans, and

tyrosine kinases play critical roles in regulating these processes.1-3 The Src-family tyrosine

kinases (SFKs) have been implicated in promoting differentiation in a number of cell

types such as skin and lens keratinocytes, oligodendrocytes, and endometrial cells. 3-8 In

addition, increased activity of the SFKs has been associated with a variety of epithelial

tumors including colonic adenocarcinoma, mammary carcinoma and murine cutaneous

squamous cell carcinoma.9-13 Given the importance of SFKs in cellular physiology, cells

have evolved a variety of mechanisms to regulate the ir activity.

Structural analyses have shown that SFKs can form an inactive “closed”

configuration, with the SH2 domain bound to the phosphorylated C-terminal tyrosine

(Y527) and the SH3 domain associated with a polyproline motif in the linker region; this

configuration prevents phosphorylation at tyrosine 416 in the activation loop rendering the

kinase inactive.14, 15 Given these structural data, it has been hypothesized that molecules

containing ligands for the SH2 and SH3 domains of SFKs may disrupt the SH2-dependent

and SH3-dependent intramolecular interactions and promote opening of the “closed”

configuration, phosphorylation of tyrosine 416, and activation of the kinase.14, 15

Theoretically, if a molecule contained a stronger ligand for the SFK SH2 domain than the

C-terminal pY527 motif, then such a molecule could activate Src kinases regardless of

Csk activity (the kinase which induces Y527 phosphorylation), thereby independently

promoting important regulatory signals.2, 16 Some SFK-activating molecules have been

identified, including FAK and Sin; however, these molecules do not contain the highest

affinity ligands for the SFK SH2 domain, a pYEEI motif.17-19


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The Src-activating and signaling molecule (Srcasm) is a recently described

activator and substrate of SFKs that contains the optimal ligands for the SH2 and SH3

domains of SFKs, and thus can activate SFKs in vitro.20 In addition, phosphorylated

Srcasm can associate in vitro with Grb2 and p85 (the regulatory subunit of PI-3 kinase),

molecules that regulate important signaling pathways involving RAS-MAP kinases and

Akt respectively.19-21 Given the molecular attributes of Srcasm, we speculated that it may

represent an important regulator of SFKs in cells.

In this report, we provide evidence to support such a role of Srcasm in

keratinocytes. Srcasm is an SFK substrate downstream of the EGF receptor, and it

activates endogenous cellular SFKs with a preference for Fyn and Src. Tyrosine

phosphorylation of Srcasm is dependent on the presence of EGFR ligands, EGFR activity,

and SFK activity. In addition, Srcasm appears to modulate p44/42 MAP kinase activity in

a manner dependent on EGF stimulation. Srcasm also promotes downstream signaling

events such as stimulation of Elk-1-dependent transcription, and it decreases the S-phase

fraction of keratinocytes under a variety of conditions. Increased levels of Srcasm

promoted expression of differentiation markers in primary keratinocytes, and Srcasm

protein levels were found to be decreased in lesions of keratinocytic neoplasia. The data

presented provide a molecular context for Srcasm signaling in keratinocytes and suggest

that Srcasm may play an important role regulating SFKs and differentiation in epithelial

cells.


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Experimental Procedures:

Adenovirus Construction: Srcasm adenovirus (Ad-Srcasm) was made by the adenovirus

vector core at the University of Pennsylvania by cloning hemagluttinin- tagged Srcasm

(HA-Srcasm) into a shuttle vector containing a CMV promoter. The expression cassette

was excised and ligated into an adenoviral backbone vector (pAdX) and the intactness of

the coding region was performed by restriction analysis. DNA was amplified and

linearized with Pac I, then transfected into 293 cells. Viral induced cytopathic change

was confirmed microscopically, and the virus was amplified then purified by CsCl

gradient centrifugation. The control adenovirus (Ad-Con) contains the GFP coding

region driven by a bacterial LacZ promoter. Hence, it expresses only in bacterial cells,

and serves as a control for adenoviral infection.

Primary human keratinocyte culture, Infections/Transfections, and pharmacologic

manipulations:

Cultures of neonatal human keratinocytes were obtained from foreskins. After

isolation, the cells were cultured in MCDB-153 medium, supplemented with 0.1mM

ethanolamine(Sigma), 0.1mM O-phosphoethanolamine(Sigma), 10µg/L hEGF (

Invitrogen), 5x10-7 M hydrocortisone(Sigma), 5mg/L insulin (Sigma), bovine pituitary

extract BPE (150 µg/ml), 100 units/ml penicillin and 100 mg/L Streptomycin, 70µM

calcium, and maintained at 37 °C with an atmosphere of 5% CO2. Growth factor

depletion was conducted in MCDB base lacking insulin, EGF, and BPE. All

keratinocytes analyzed were less than passage 4.


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Keratinocytes at approximately 60-70% confluence were infected with Ad-

Srcasm or Ad-Con at the indicated MOI under the culture conditions described above,

and usually were analyzed or manipulated 16 hours after infection.

Luciferase assays utilized the Pathdetect Elk-1 trans-reporting assay (Stratagene)

according to the manufacturer’s recommendations. Cells were transfected in MCDB (w/o

P/S) using a ratio of 1µg of DNA to 5 µl of Lipofectamine (Invitrogen) according to the

manufacturer’s instruction. Cells were allowed to recover for 18 hours before virus

infection and subsequent experimentation.

Prior to EGF, TGF-α, KGF stimulation, cells were deprived of growth factors for

24h, then incubated with hEGF (10 or 100 ng/ml, Invitrogen), hTGF-α ( 0.1 ng/ml,

Sigma #T7924), hKGF (10 ng/ml, Research Diagnostics Inc.) for the indicated times.

Some cells were incubated with Src-family kinase selective inhibitor PP2 (10 µM), or its

negative control PP3 (10 µM), or specific EGFR kinase inhibitor AG112 (20 µM) or its

negative control AG9 (20 µM), or the MEK 1/2 inhibitors U0126 (10µM) or its negative

control U0124 (10µM) 60min before cell lysis (all from Calbiochem, La Jolla, CA).

Antibodies: α-phosphotyrosine (Upstate Biotechnology, 4G10) was used at 1/1000 for

western blotting. α-phosphotyrosine immunoprecipitation of endogenous Srcasm was

performed overnight at 4oC using 1.5 mg of protein lysate and 2.5 µg of antibody; α-

phosphotyrosine immunoprecipitation for transduced Srcasm was performed using 0.5

mg of lysate for 15 minutes with 1.5 µg of antibody using the Catch and Release

Reversible Immunoprecipitation system (Upstate Biotechnology). High affinity α-HA

antibody (clone 3F10, Boehringer Mannheim) was used at 1/1000 for western blotting


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and at 2 µg/ml for immunoprecipitations. α−activated Src family kinase antibody

(phosphor-Tyr416) (Cell Signaling) was used at 1/1000 for western blot. α-p44/42 MAP

kinase antibody and α-phospho-p44/42 MAP kinase antibody (Cell Signaling) were used

at 1/1000 to detect total or phosphorylated p44/42 MAP kinase. α-Fyn (SC-16), α-Src

(SC-19), and α-Yes (SC-14, all from Santa Cruz) were all used at 1:1000 for western

blotting and 3 µg was used for immunoprecipitation. α-Src2 antibody (SC-8056) that

detects Src, Fyn, and Yes was used at 1:500. α-Srcasm antibody is a polyclonal antibody

that was generated by incubating rabbits with three purified GST fusion proteins

spanning murine Srcasm (aa 1-200, aa 150-400, aa 389-474). For western blotting, the

Srcasm antisera is diluted 1:1000. For endogenous Srcasm immunoprecipitation, IgG

was purified from the crude serum via Protein A affinity chromatography; 6 µg of this

IgG fraction was used for each immunoprecipitation. The specificity of the α-Srcasm

antibody was demonstrated by western blotting COS protein lysates overexpressing

Srcasm; preincubating the antisera with the 1µM Srcasm fusion proteins for 30 minutes at

room temperature ablates detection of Srcasm (Fig 8A).

Immunoblotting and Immunoprecipitation: Cell lysates were prepared using RIPA

lysis buffer (150 mM NaCl, 50mM Tris HCl pH 8.0,1% Triton X-100, 0.1%SDS,

0.5%DOC, 5 mM EDTA, 10 mM NaF, 10 mM sodium pyrophosphate, 1 µg/ml

Aprotinin, 100µM Leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1mM NaVO4).

Tissue samples of normal skin and squamous cell carcinoma tissue were obtained after

Moh’s surgery and frozen at -80oC until use (IRB protocols # 707777 and 706409). The

tissue samples were diced and homongenized in RIPA buffer. The lysates were incubated

on ice for 10 minutes and cleared by centrifugation at 14,000 x g for 10 minutes at 4oC.


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The supernatants were collected and assayed for protein content using the MicroBCA

protein assay kit (Pierce Chemical Co.). For IP with α-HA antibody, typically 1 mg of

pre-cleared lysates were sequentially incubated with α-HA, Rabbit α-Rat IgG and protein

A agarose. IP with α-pY antibody was carried out with the Catch and Release reversible

immunoprecipitation system (Upstate). For α-Srcasm immunoprecipitation of Srcasm,

typically 1.5 mg of keratinocyte lysate were utilized, and the immunoprecipitations were

carried out for approximately 16 hours. Equal amounts of lysates or washed

immunoprecipitates were separated by SDS-PAGE and transferred to PolyScreen PVDF

transfer membrane (PerkinElmerLife Sciences, Inc.). For filaggrin detection, Western

blots were conducted in a standard manner with the indicated antibodies and developed

using an enhanced chemiluminescence kit as described by the manufacturer (Lumilight

Plus, Boehringer Mannheim).

Immunofluorescence analysis: Cells were cultured in 2-well Lab-Tek chamber slides

(Nalge Nunc International) and infected with Ad-Srcasm or Ad-Con (MOI-200). Cells

were fixed with ice cold 4% paraformaldehyde in PBS for 10 min on ice then

permeabilized with 0.2% triton/PBS for 10 min. Fixed cells were washed with PBS and

then incubated with primary antibodies for 60 minutes at room temperature, followed by

washing, and incubation with the appropriate secondary antibodies in the same manner.

Primary antibodies used at a 1:100 dilution include: phospho-Y416 (Cell Signaling

Technology), phospho p44/42 (Cell Signaling Technology), HA-high affinity (Roche),

Src ( SC-19, Santa Cruz), Fyn (SC-16, Santa Cruz), Yes (SC-14, Santa Cruz). α-

Filaggrin (PRB-417P, Covance) was used at 1:200. α-BrdU (#1299964, Roche) was

used at 1:50. Secondary antibodies were species-specific for the primary and FITC or

Texas Red conjugated; these antibodies were used at 1:100 (Jackson ImmunoResearch).


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Nuclei were counterstained using DAPI. Imaging was performed on an IX-81 inverted

fluorescence scope under oil coupled with a Cooke Sensicam digital camera. Where

indicated, deconvolved, confocal images (nearest neighbors algorithm) and image

overlay were performed using Slidebook, version 4. Magnification-600X

In vitro kinase Assays: PHKs were infected with Ad-Con or Ad-Srcasm at MOI-200 and

cultured in MCDB complete media. Sixteen hours post- infection, cells were lysed in

RIPA buffer, and 1 mg of cell lysate was subjected to immunoprecipitation with 3 µg of

α-Fyn, α-Src, or α-Yes for 16 hours at 4oC. The immunoprecipitated Fyn, Src, and Yes

were subjected to in vitro kinase assays according to manufacturer’s specifications (Src

Assay kit #17-131, Upstate Biotechnology). Phosphocellulose filters with bound 32P –

labeled substrate were assessed by liquid scintillation using Econofluor-2 (Packard

Instrument Company, Cat# 6NE9699) in a Beckman LS-. Background CPM activity

(from a no kinase control reaction) was subtracted from all values. CPM values of assays

of SFKs from control cells was set = 1; values of assays from Ad-Srcasm cells were

divided by the corresponding value from control infected cells to obtain fold-stimulation.

Data presented from 2 independent experiments.

Luciferase Assays: Human primary keratinocytes were plated in complete MCDB media,

allowed to reach 50% confluency, and the transfected with plasmids from the Pathdetect

trans-reporting system (Stratagene), specific for assaying Elk-1 dependent transcription

from serum-response elements. Primary keratinocytes plated at 5 X 104 per 35 mm well

were transfected using Lipofectamine (5µl/µg DNA) with 0.5 µg οf pFA2-Elk-1 and 1.0

µg of pFR-Luc. Approximately 16 hours after transfection, cells were not infected or

infected with Ad-Srcasm or Ad-Con at an MOI of 200. After 18 hours of infection, some

cells were stimulated for 5 h with 100 µg/L hEGF. Luciferase activity in equivalent


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amounts of cell lysates was determined using the firefly luciferase assay system

(Promega ) in a Monolight 3096 luminometer (Becton-Dickinson) equipped with

Simplicity 2.0 software. Relative luciferase units reflect the numerical values obtained

from the luminometer; values represent the averages of three experiments with the

standard deviation indicated by bars. Equivalent amounts of cell lysate were subjected to

western blotting to detect Srcasm.

Flow cytometry proliferation assay: Sub-confluent PHKs infected with Ad-Srcasm or

Ad-Con (MOI-200) 18 hours prior were labeled with 10µM BrdU for 3h under standard

MCDB culture conditions (see above). Then, cells were harvested and stained with FITC

conjugated anti-BrdU antibody and 7-AAD following the BrdU flow kit instruction (BD

Pharmingen); the analysis parameters used are those recommended by the manufacturer.

The stained cells were analyzed by FACScan machine (BD Pharmingen); the cell

populations were sorted into G0/G1 (gate R2), G2/M (gate R3), and S phase (gate R4)

according to the manufacturer’s specifications. In figure 6C, subconfluent PHKs were

infected as above, deprived of growth factors for 16 hours, and then some cells were

stimulated with EGF (10 ng/ml) for the indicated times. These cells were collected,

washed in PBS, and fixed in 70% ethanol, overnight at 4oC. Cells were pelleted then

washed in PBS with 2% FCS, and subjected to passage through a 0.2 µm filter. Cells

were treated with RNase A and stained with propidium iodide. Cells were analyzed on a

FACScan machine using ModFit LT 3.1 (Verity Software House) to determine S-phase

fraction. Data representative of two experiments.

BrdU labeling and filaggrin blotting: Cells were cultured in 2-well Lab-Tek chamber

slides (Nalge Nunc International) and infected with Ad-Srcasm or Ad-Con (MOI-200) in


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MCDB complete media. 24 hours after infection, cells were labeled with BrdU (10 µM)

for 3 hours and then fixed and permeabilized with 50 mM glycine, 70% ethanol, pH=2

for 20 minutes at 4oC. Cells were stained for BrdU, filaggrin, or Srcasm as indicated

under Immunoflourescence Analysis. Cells stained for filaggrin and Srcasm were fixed

and permeabilized as indicated under Immunofluorescence Analysis.

For filaggrin western blotting, PHKs were plated on collagen coated dishes and

infected with Ad-Srcasm or Ad-Con (MOI-200). Cells were cultured in MCDB complete

media. At the times indicated (post- infection), cells were lysed in RIPA and equivalent

protein amounts were subjected to western blot analysis

Immunohistochemical detection of Srcasm in formalin-fixed biopsy specimens:

Formalin-fixed, paraffin-embedded tissue samples of actinic keratosis, squamous cell

carcinoma – in situ, invasive squamous cell carcinoma and unremarkable skin were

collected from the dermatopathology archives of the University of Pennsylvania

Department of Dermatology with IRB approval under protocol 704450. Biopsy

specimens containing unremarkable epidermis adjacent to lesional skin were selected as

normal skin provides an internal control. For immunohistochemical staining, tissue

samples were blocked for 1 hour at room temperature with 10% horse serum, then

incubated for 1 hour with affinity-purified rabbit polyclonal anti-Srcasm antibody

(4µg/ml). The antibody was affinity purified by incubating anti-sera with GST

immobilized on PVDF membranes followed by the Srcasm fusion proteins immobilized

on PVDF membranes. The anti-Srcasm antibody was eluted and handled as previously

discussed.22 Control preimmune IgG was purified using protein A agarose and used at

the same concentration as the affinity purified antibody. Immunostaining was performed


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using the avidin-biotin complex technique (Roche). Increases or decreases in expression

were made by comparing lesions with adjacent normal skin from the same tissue section

and with normal skin from separate biopsies.

Statistical Analysis: Values are expressed as the average ± SD of the indicated number

of samples for each experiment. Student’s T-test was used to compare data between two

groups. P< 0.05 was considered statistically significant.

Results:

EGFR ligands and SFKs promote tyrosine phosphorylation of Srcasm in

primary human keratinocytes. Since EGF stimulation of cells can activate Src kinases,

the effect of EGF on the tyrosine phosphorylation of endogenous Srcasm was evaluated in

primary human keratinocytes (Fig. 1) 23, 24 Immunoprecipitation with α-Srcasm followed

by western blotting with α-phosphotyrosine demonstrated increased levels of Srcasm

tyrosine phosphorylation in EGF-treated keratinocytes (Fig. 1A). Similarly,

immunoprecipitation with α-phosphotyrosine from EGF-treated cells followed by western

blotting using α-Srcasm demonstrated more phospho-Srcasm present in EGF-treated cells

(Fig. 1B). Parallel experiments with TGF-α and KGF were performed; TGF-α treatment

of keratinocytes increased tyrosine phosphorylation of Srcasm while KGF treatment did

not (Fig. 1B). These results show that EGF and TGF-α, both EGFR ligands, induce

tyrosine phosphorylation of endogenous Srcasm.


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The behavior of adenovirally-transduced HA-Srcasm was examined in PHKs.

PHKs transduced with HA-Srcasm adenovirus and cultured in complete media exhibited

readily detectable levels of tyrosine-phosphorylated Srcasm (Fig 1C), while PHKs

deprived of growth factors (EGF, insulin, and bovine pituitary extract) for 24 hours

contained very low levels of tyrosine phosphorylated Srcasm. PHKs stimulated with EGF

demonstrated a rapid and significant increase in tyrosine-phosphorylated Srcasm (Fig.

1C); these results for transduced Srcasm parallel those for endogenous Srcasm.

Upstream signaling events necessary for tyrosine phosphorylation of Srcasm were

explored using pharmacologic inhibitors of EGFR and SFKs. Treatment of keratinocytes

with inhibitors of SFKs (PP2) and EGFR (AG112) ablated the EGF-induced tyrosine

phosphorylation of Srcasm (Fig. 1D), demonstrating tha t EGF-induced tyrosine

phosphorylation of Srcasm requires the activity of both EGFR and SFKs. In addition,

these data show that adenoviral HA-Srcasm appears to be a useful model for

characterizing the effect of increased Srcasm levels on keratinocyte signaling.

Srcasm promotes cellular tyrosine phosphorylation and activates SFKs in

keratinocytes. EGF-treatment and elevated Srcasm levels correlate with increased

tyrosine phosphorylation of a number of cellular proteins (Fig. 2A). Notably, a doublet at

kDa show increased tyrosine phosphorylation with EGF treatment or increased Srcasm

levels.

Since Srcasm can activate SFKs in vitro, the effect of Srcasm on endogenous

keratinocyte SFK activation was evaluated. In non- infected PHKs, the levels of activated

SFKs were low (Fig. 2B); similar results were seen with control adenovirus. In contrast,

infection of the keratinocytes with Ad-Srcasm led to significantly higher levels of

activated SFKs (Fig. 2B). Increased levels of Srcasm also potentiated the ability of EGF


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to activate Src-kinases (Fig. 2B). These results show that increased levels of Srcasm are

associated with activation of endogenous SFKs in PHKs, and that increased Srcasm level

can lead to higher levels of activated SFKs secondary to EGF treatment.

The ability of Srcasm to activate SFKs is dose-dependent; the level of activated

SFKs is proportional to the level of cellular Srcasm (Fig 2C). Association of increased

Srcasm levels with SFK activation was evaluated at the cellular level via

immunofluorescence. Cells containing higher amounts of Srcasm have higher levels of

activated SFKs (Fig. 2D).

Srcasm differentially activates SFKs in primary keratinocytes. Since

keratinocytes express Fyn, Src, and Yes; the effect of increased Srcasm levels on each

SFK family member was evaluated. Immunoprecip itation of Fyn from keratinocytes

containing higher levels of Srcasm followed by western blotting to assess levels of

activated kinase demonstrated increased Fyn activation when compared to control cells

(Fig. 3A). Activation of Src was also seen in similar experiments. Interestingly, Yes

showed decreased activation in Ad-Srcasm infected cells relative to controls (Fig 3A).

The effect of increased Srcasm levels on SFK activity was also evaluated using in

vitro kinase assays. Both Fyn and Src showed increased kinase activity in PHKs infected

with Ad-Srcasm compared to control cells (Fig. 3B). The kinase activity of Yes was

mildly decreased in Ad-Srcasm infected PHKs compared to control cells (Fig. 3B). Using

two different experimental methods, increased Srcasm levels are associated with increased

Fyn and Src activity but with decreased Yes activity.

Immunofluorescence studies demonstrated more prevalent co- localization of Fyn

and Src with Srcasm, as indicated by increased yellow color in the merged confocal

images (Fig. 3C). In contrast, relatively little co- localization of Yes and Srcasm was seen


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in similar experiments (Fig. 3C). Therefore, the degree of cellular co-localization between

Srcasm and Fyn, Src, or Yes appears to correlate with the ability of Srcasm to activate

these kinases.

Srcasm modulates p44/42 MAP kinase activation in primary keratinocytes.

EGF stimulation of primary human keratinocytes leads to the activation of ERK2 kinase

(p44 MAP kinase) via Ras.25 Given that Srcasm can amplify some EGF-dependent

signals, the effect of Srcasm on p44/42 MAP kinase activa tion was evaluated in primary

keratinocytes.23 Keratinocytes with increased Srcasm levels demonstrated a significant

increase in basal p44/42 MAP kinase activity when compared to control cells (Fig 4A). In

fact, the level of p44/42 MAP kinase activation in Ad-Srcasm cells mirrors that seen in

EGF-stimulated control cells (Fig. 4A). At early time points of EGF stimulation,

keratinocytes with elevated levels of Srcasm maintained a high level of p44/42 MAP

kinase activation similar to that in control cells, confirming that the level of p44/42 MAP

kinase activation already was at near-maximal levels in the unstimulated Ad-Srcasm

infected cells. However, by ten minutes, EGF stimulation led to a marked decrease in

p44/42 activation in cells with high levels of Srcasm, whereas levels of activated p44/42

were elevated and stable in control cells. After 30 minutes of EGF stimulation, the levels

of p44/42 activation dropped in control cells (data not shown). Based on these

observations, increased Srcasm levels activate p44/42 MAP kinases in unstimulated

keratinocytes, and, in the same cells, EGF-stimulation promotes a rapid deactivation of

these kinases. Therefore, Srcasm has the ability to modulate p44/42 MAP kinase activity

in keratinocytes.

The Srcasm-dependent activation of p44/42 MAP kinases was investigated using

pharmacologic inhibitors of EGFR, SFKs, and MEK 1/2. Preincubation of keratinocytes


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with AG112, an EGFR kinase inhibitor, resulted in a decrease in the Srcasm-dependent

activation of p44/42 MAP kinases (Fig. 4B). In a similar manner, preincubation of

keratinocytes with PP2, an inhibitor of SFKs, and UO126, a MEK 1/2 kinase inhibitor,

ablated the Srcasm-dependent activation of p44/42 MAP kinases. These results

demonstrate that Srcasm media tes p44/42 MAP kinase activation through a pathway

dependent on the activity of EGFR, SFKs, and MEK 1/2.

The relationship between Srcasm levels, p44/42 MAP kinase activation, and EGF

treatment was investigated at the single cell level using immunofluorescence.

Keratinocytes infected with control adenovirus demonstrated very weak staining for

activated p44/42 MAP kinase (Fig. 4C), while control cells stimulated with EGF exhibited

stronger staining for activated p44/42 MAP kinase. In contrast, cells with higher levels of

Srcasm demonstrated markedly increased staining for activated p44/42 MAP kinase prior

to stimulation. EGF treatment of these cells led to decreased levels of activated p44/42

MAP kinase; however, in cells with lower levels of Srcasm staining, EGF induced high

levels of activated p44/42 MAP kinase staining (Fig. 4C). Prominent nuclear localization

of activated p44/42 MAP kinase was seen in keratinocytes over expressing Srcasm prior to

EGF stimulation or in control cells stimulated with EGF (Fig. 4C, lower panel). EGF

treatment of Ad-Srcasm cells resulted in a marked decrease of activated p44/42 MAP

kinase in the nucleus. The results of the immunofluorscence studies confirm at the single

cell level that Srcasm stimulates p44/42 activity under basal conditions and promotes

rapid deactivation of these kinases secondary to EGF stimulation. Based on these data,

Srcasm transmits signals from EGFR and SFKs that can regulate the activity of p44/42

MAP kinases.


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Srcasm promotes Elk-1-dependent transcription. Elk-1 is a transcription

factor of the ETS family that can be phosphorylated by kinases including activated

p44/42 MAP kinases.26 Subsequently, phosphorylated Elk-1 can promote transcription

from serum-response elements.27-29 Because Srcasm can activate p44/42 MAP kinases,

its ability to promote Elk-1 dependent transcription was evaluated in primary

keratinocytes. In this transcriptional assay, preliminary experiments demonstrated that 5

hours of EGF stimulation resulted in consistent EGF-inducible luciferase activity, and

that EGF stimulation for five hours resulted in higher levels of p44/42 activation in Ad-

Srcasm infected PHKs relative to control cells (data no t shown). Uninfected

keratinocytes transfected with the Elk-1 plasmid and reporter plasmid demonstrated only

low levels of luciferase activity basally or after EGF stimulation (Fig. 5). Infection of

keratinocytes with control adenovirus increased luciferase activity compared to

uninfected cells under both conditions. Cells infected with Ad-Srcasm demonstrated the

highest levels of luciferase activity, and the EGF-dependent induction of lucifease

activity in these cells was significantly greater than in uninfected cells or control infected

cells. These data suggest that Srcasm can promote transcriptional events downstream of

the EGF-RAS-MAP kinase pathway involving Elk-1, and Srcasm may potentiate the

effect of EGF on Elk-1 dependent transcription in primary keratiocytes.

Increased Srcasm expression in keratinocytes inhibits DNA synthesis and

promotes differentiation. Increased Fyn activity in primary murine keratinocytes

inhibits cell proliferation and promotes the expression of differentiation markers such as

transglutaminase and fillagrin.6, 30 Because Srcasm can promote activation of SFKs in

human keratinocytes, including Fyn, the effect of Srcasm on cell proliferation and

differentiation was evaluated. Keratinocytes infected with control or Srcasm adenovirus


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were labeled with BrdU and 7-AAD then subjected to FACS analysis to determine the S-

phase fraction. In MCDB complete media, approximately 29% of control keratinocytes

are in S-phase; however, keratinocytes with increased levels of Srcasm demonstrate only

20% of cells in S-phase (Fig. 6A and B).

The ability of increased Srcasm levels to decrease the S-phase fraction of PHKs

was tested under a variety of growth conditions. Under both growth factor depletion and

EGF stimulation, Ad-Srcasm infected PHKs had significantly lower S-phase fractions than

control cells (Fig. 6C). These data confirm that Ad-Srcasm infected PHKs have a

decreased S-phase fraction compared to control cells.

Decreases in the percentage of keratinocytes in S-phase could correlate with

initiation of differentiation; therefore, the effect of increased Srcasm on keratinocyte

differentiation was assessed. The effect of increased Srcasm on filaggrin expression and

BrdU labeling was characterized using immunofluorescence. Immunofluorscence staining

for BrdU and Srcasm demonstrated that keratinocytes with higher levels of Srcasm did not

contain nuclear BrdU, while cells with lower levels of Srcasm had nuclear BrdU (Fig. 7A,

left panel). In similar experiments, keratinocytes expressing the filaggrin precursor

protein did not demonstrate nuclear staining for BrdU, while cells with little filaggrin

staining did (Fig. 7A, middle panel). Keratinocytes with increased Srcasm levels

demonstrated higher levels of the filaggrin precursor protein (Fig. 7A, right panel).

Therefore, increased Srcasm levels correlate with decreased BrdU labeling and increased

filaggrin expression in keratinocytes.

Western blotting of lysates from keratinocytes infected with the Srcasm

adenovirus demonstrated increased levels of filaggrin at 3 and 5 days post- infection

relative to control cells (Fig. 7B). Interestingly, the levels of cellular Srcasm also


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increased in a parallel manner (Fig 7B). Together, these data demonstrate that increased

Srcasm levels inhibit keratinocyte proliferation and promote aspects of the keratinocyte

differentiation program such as increased filaggrin expression.

Srcasm protein levels decrease in human cutaneous neoplasia. Since increased

levels of Srcasm inhibit cell proliferation and promotes keratinocyte differentiation

program, the level of Srcasm was evaluated in human cutaneous neoplasia where

keratinocyte differentiation is disrupted. This question was addressed by

immunohistochemistry using affinity-purified rabbit polyclonal α-Srcasm on sections

representing the various stages of human cutaneous neoplasia and by wesetern blotting of

lysates from tissue samples.31 The specificity of α-Srcasm was demonstrated by detecting

increased levels of Srcasm in COS cell lysates overexpressing Srcasm, and blocking

detection by preincubating the antisera with Srcasm fusion proteins (Fig. 8A). Staining

with purified IgG from pre-immune serum (Fig. 8B) or secondary antibody alone (data not

shown) did not demonstrate significant specific epidermal staining. Staining with pre-

immune IgG did demonstrate non-specific staining of the stratum corneum, which is

commonly seen but was not seen with the affinity purified antibody (Figs. 8C-F).

A total of seventeen actinic keratoses (early dysplasia), eight squamous cell

carcinomas in-situ (SCIS), and twelve squamous cell carcinoma (SCC) biopsy samples

were examined. For each experiment, two biopsies of normal skin were used as controls.

In addition, nearly all lesional samples examined contained adjacent portions of normal

epidermis on the same tissue profile which served as internal controls. When compared

to normal skin samples and perilesional skin, there were readily detectable decreases in

Srcasm staining in 15/17 actinic keratoses, 7/8 SCC-IS, and 10/12 invasive SCC (Figs. 8


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C-E). Those lesions that did not show a decrease in Srcasm staining showed similar

levels of Srcasm as did the controls (data not shown). The relative decrease of Srcasm

staining between lesional and normal epidermis was more readily detected in SCCs than

in SCC-IS and actinic keratoses; however, this difference canno t be quantified using

immunohistochemistry. In addition, lysates derived from cutaneous SCC contain much

lower amounts of Srcasm than lysates from unremarkable skin (Fig. 8G). These data

suggest that decreased Srcasm levels are associated with cutaneous keratinocytic

neoplasia.

Discussion:

This manuscript provides novel data regarding how Srcasm influences important

regulatory signaling pathways involving the EGFR, SFKs, and p44/42 MAP kinases, and

it provides insight into how Srcasm may affect the biology of primary human

keratinocytes. Previous work has shown that Srcasm can activate SFKs and that it is an in

vitro substrate of these kinases.20 Once tyrosine phosphorylated, Srcasm can associate

with important cell signaling molecules including: SFKs, Grb2, or p85, the regulatory

subunit of PI-3 kinase.20 Given these characteristics, Srcasm may activate SFKs and help

transmit signals from these kinases to other intracellular signaling pathways. Until now, it

was not known what signaling pathways are influenced by Srcasm or how Srcasm is

regulated by specific cell signaling pathways.

The EGFR signaling pathway is important for regulating cell growth and

differentiation in keratinocytes and other epithelial cells. A large body of work supports

the hyothesis that signaling through EGFR promotes keratinocyte proliferation and is

important in keratinocytic neoplasia.32-36 In addition, signaling through EGFR may also

promote differentiation in keratinocytes cultured on specific substrates.37 One


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consequence of EGFR stimulation is activation of SFKs.38 Since activation of these

kinases represents an important step in transmitting the EGF-dependent signal, it was

important to determine if Srcasm was involved with EGFR signaling in keratinocytes.39,

40 EGF and TGF-α stimulation of keratinocytes promote tyrosine phosphorylation of

Srcasm, and increased Srcasm levels are associated with higher levels of SFK activation.

Therefore, Srcasm is a component of the EGF signaling pathway that activates SFKs and

may promote SFK-dependent signaling in keratinocytes. Because tyrosine

phosphorylation of Srcasm was dependent on both EGFR and SFK activity, other

tyrosine kinases activated by EGFR, including Janus kinases, probably do not efficient ly

phosphorylate Srcasm.41

Originally, Srcasm was isolated using Fyn as the bait for a yeast two hybrid

screen of a keratinocyte library.20 Therefore, it was not surprising to find that Srcasm can

activate Fyn in keratinocytes; however, it is intriguing that Srcasm preferentially activates

Fyn and Src over Yes in human keratinocytes (Fig. 3). This observation implies that

Srcasm may preferentially promote Fyn and Src-dependent signals in keratinocytes. In

primary murine keratinocytes, enhanced Fyn activity is associated with differentiation;

increased calcium exposure, which induces differentiation, preferentially activates Fyn

but not Src or Yes.6, 42 Fyn activity is important also for proper expression of filaggrin

and transglutaminase and decreased DNA synthesis, suggesting that increased Fyn

activity is associated with keratinocyte differentiation.6, 30 It appears that increased

Srcasm levels promote human keratinocyte differentiation by decreasing DNA synthesis

and promoting filaggrin expression (Figs 6 and 7). Additional studies in other cell lines

will reveal if Srcasm can promote differentiation in a variety of cells.


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An important question in cell biology regards the means by which growth-factor

tyrosine kinases and non-receptor tyrosine kinases are linked to the p44/42 MAP kinase

pathway. In keratinocytes, activation of the EGFR has been linked to activation of p44/42

MAP kinases.43 However, the mechanism by which SFKs are integrated into the EGFR-

MAP kinase pathway has not been well defined. Srcasm appears to link SFKs to the

EGFR-MAP kinase pathway because increased Srcasm levels were associated with higher

levels of activated SFKs and p44/42 MAP kinases, and this activation was minimized by

inhibitors of EGFR and SFKs (Fig. 4). Also, the observation Srcasm-dependent activation

of p44/42 MAP kinases was abrogated by U0126, a MEK 1/2 inhibitor. Therefore,

Srcasm appears to function at an important signaling nexus linking EGFR and SFKs with

MEK 1/2 and p44/42 MAP kinases.

Modulation of p44/42 MAP kinase activity is critical for regulating cell

proliferation and differentiation.25 The ability of Srcasm to modulate p44/42 MAP kinase

activity correlates well with a role in promoting keratinocyte differentiation. The EGF-

induced inactivation of p44/42 MAP kinases in cells over-expressing Srcasm is an

interesting observation because of the difference between short-term activation and long-

term activation of this pathway. Interestingly, persistent EGF signaling which activates

p44/42 MAP kinases, can decrease proliferation of neoplastic cells whereas transient

activation of the same pathway promotes proliferation.44

To transiently deactivate p44/42 MAP kinase in the early stages of EGF

stimulation, Srcasm may activate/recruit a phosphatase, since levels of total p44/42 MAP

kinase remain stable. An alternative interpretation is that the fraction of activated p44/42

MAP kinases may be small compared to the total cellular pool of p44/42 MAP kinases;

therefore, degradation of activated p44/42 MAP kinases by a Srcasm-promoted


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proteosome-dependent mechanism cannot be completely excluded. It will be interesting

to determine the mechanism by which increased Srcasm levels promote EGF-dependent

transient deactivation of p44/42 MAP kinases and if this phenomenon occurs in

transformed cells.

Decreased Srcasm levels were seen in lesions of human keratinocytic neoplasia

(Fig. 8). This observation raises the hypothesis that Srcasm expression may be

downregulated in cutaneous neoplasia. It is well known that uncontrolled Src kinase

activity can lead to neoplasia in a variety of cell types.1 In fact, overexpression of Src in

murine keratinocytes enhances tumor promotion and malignant progression.45 One

hypothesis stemming from these observations is that Srcasm levels may be important for

directing SFK signaling through the proper pathways. Recent studies have shown that the

GAT domain of Srcasm and TOM1 may play a role in binding ubiquinated proteins and

promoting ubiquination.46-48 It will be important to determine how the GAT domain of

Srcasm regulates SFK function. Further studies, including the use of transgenic mice

overexpressing Src kinases and Srcasm, will clarify the roles of these molecules in

cutaneous neoplasia.

The data presented begin to construct a signaling paradigm for Srcasm in which

this molecule transmits signals from the EGFR and SFKs to important cell regulatory

signaling pathways (Fig. 9). Srcasm activates the p44/42 MAP kinase/Elk-1 pathway in a

manner dependent on the activity of the EGF receptor, SFKs, and MEK 1/2 kinases.

Therefore, Srcasm appears to modulate a key growth regulatory signaling pathway in

keratinocytes. Additional work to characterize the role of Srcasm in modulating the EGF-

RAS-MAP kinase pathway may provide new insights on how this critical pathway can be

regulated. Genetically engineered mice overexpressing or lacking SFKs and Srcasm


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should provide novel insights about Srcasm function and how it mediates aspects of SFK

signaling.

Acknowledgements: The authors thank Drs. John Stanley and Gary Koretzky for helpful discussions and support. The authors also thank Dr. Paul Stein for assistance with anti-Srcasm antibody production. This work was supported, in part, by the University of Pennsylvania and the Department of Dermatology, University of Pennsylvania Medical School. In addition, support was providec by NIH grants (KO8-AR47597, NCI P01-CA098101) and a Career Development Award from the Dermatolgy Foundation to JTS.

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Figure 1. Tyrosine phosphorylation of Srcasm is promoted by EGFR ligands and

SFKs. (A and B) EGF and TGF-α promote tyrosine phosphorylation of endogenous

Srcasm. Primary human keratinocytes (PHKs) were depleted of growth factors fo r 24

hours then stimulated with 10 ng/ml EGF, 0.1ng/ml TGF-α, or 10 ng/ml KGF for 5

minutes. Cell lysates were immunoprecipitated with α-Srcasm or α-phosphotyrosine;

immunoprecipitates were analyzed by western blotting with α-phosphotyrosine or α-

Srcasm. Apparent molecular weight of endogenous Srcasm- 58-60 kDa. N=2 WCL-

whole cell lysate. N=2 (C) Phosphorylation of transduced HA-Srcasm is EGF dependent.

PHKs were infected with Ad-Con or Ad-Srcasm (MOI of 50 or 200), and cultured in

MCDB with 10 ng/ml EGF. Cells were deprived of growth factors for 24 hours (SS), and

then stimulated with EGF 10 ng/ml for 5 minutes. Lysates were immunoprecipitated

with α-HA, and immunoprecipitates were split then analyzed by western blotting with α-

pY or α-Srcasm. Apparent molecular weight of transduced HA-Srcasm = 60-64 kDa.


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N=2 (D) PHKs were infected with Ad-Srcasm at an MOI of 200. Cells were deprived of

growth factors for 24 hours, and then stimulated with EGF 10 ng/ml for 5 minutes. Some

cells were pre-treated with a Src-family tyrosine kinase inhibitor, PP2 (10 µM) or its

negative control, PP3 (10 µM), an EGFR tyrosine kinase inhibitor, AG112 (20 µM) or its

negative control, AG9 (20 µM) for 30 minutes before EGF stimulation. Lysates were

subjected to immunoprecipitation with α-HA; immunoprecipitates were analyzed as in D.

Figure 2. Srcasm increases cellular tyrosine phosphorylation and activates SFKs in

keratinocytes A) EGF-treatment and Srcasm overexpression increase protein tyrosine

phosphorylation. PHKs infected with Ad-Con or Ad-Srcasm (MOI-200) were subjected

to EGF stimulation. Lysates were analyzed by western blotting with α-phosphotyrosine,

α-Srcasm, or α-βactin (loading control) N=2. B) Srcasm activates endogenous SFKs.

PHKs were subjected to no virus, control adenovirus, or Srcasm adenovirus at a MOI of

200. Some cells were treated with hEGF, 100 ng/ml for 15 minutes. Cell lysates were

subjected to SDS-PAGE followed by western blotting with the following antibodies: α-

activated src kinase (pY416), α-SFK (Src2), and α-Srcasm. Data representative of four

experiments. C) Srcasm activates endogenous SFKs in a dose-dependent manner. PHKs

were cultured in MCDB complete media and infected with Ad-Con or Ad-Srcasm at the


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indicated MOI. Cell lysates were analyzed as in B. N=2 D.) Association of activated

SFKs and Srcasm by immunofluorescence. PHKs cultured in 2-well chamber slides were

infected with Ad-Con or Ad-Srcasm at a MOI of 200. Some cells were treated with EGF,

100ng/ml for 5 minutes. Cells were double-stained α-p416 (green) and α-HA (red).

DAPI was used for nuclear staining. 600X.


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Figure 3. Srcasm differentially activates SFKs. (A) Differential activation of SFKs by

Srcasm in PHKs. PHKs were infected with Ad-Con or Ad-Srcasm at an MOI of 200.

Cellular SFKs were immunoprecipitated with specific antibodies to Fyn, Src, or Yes.

The immunoprecipitates were split, subjected to SDS-PAGE, followed by western

blotting with α-pY416 and α-SFK. Data representative of 2 experiments. (B) In vitro

kinase assays show Srcasm preferentially activates Fyn and Src. Fyn, Src, and Yes were

immunoprecipitated from PHKs transduced with Ad-Con or Ad-Srcasm (MOI-200).

Immunoprecipitates were subjected to an in vitro kinase assay (see Experiment

Procedures). Fold stimulation of Fyn, Src, or Yes kinase activity from Ad-Srcasm cells

relative to control cells is shown. N=2 (C) Srcasm preferentially localizes with Fyn and

Src by immunoflurorescence. PHKs were cultured in 2-well chamber slides and infected

as in panel A. Cells were double-stained to detect Srcasm (red) and Fyn, Src or Yes

(green). DAPI was used for nuclear staining. 600X


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Fig. 4. Srcasm modulates p44/42 MAP kinase activation in keratinocytes. (A)

Primary keratinocytes were cultured in MCDB with additives (see Materials and

Methods) and then stimulated with 100 ng/ml EGF for the indicated times. Cell lysates

were subjected to western blotting with the following antibodies: activated p44/42,

p44/42, and Srcasm. N=3 (B) Srcasm-dependent activation of p44/42 MAP kinase

neccesitates activity of EGFr, SFKs, and MEK 1/2. Primary keratinocytes were infected

with Ad-Con or Ad-Srcasm at a MOI of 200. After deprivation of growth factors for 24

hours, cells were stimulated with 10 ng/ml EGF for 5 minutes. Some cells were pre-

treated with PP2 (10 µM), or PP3 (10 µM), or AG112 (20µM), or AG9 (20µM), U0126

(10µM) or U0124 (10µM) for 30 minutes before EGF stimulation. Cell lysates were

subjected to western blotting with the following antibodies: activated p44/42, p44/42,

and Srcasm. N=2 (C) Upper panel- increased Srcasm expression correlates with activated

p44/42 under culture conditions but not with short-term EGF stimulation. Primary

keratinocytes cultured in chamber slides were infected with Ad-Con or Ad-Srcasm at a

MOI of 200. Some cells were treated with EGF, 100 ng/ml for 5 minutes. Cells were

double-stained to detect phosphorylated p44/42 (green) and HA-tagged Srcasm (red).

DAPI was used for nuclear staning. Lower panel, as above but with no DAPI staining to

highlight nuclear localization of activated p44/42 MAP kinases. 600X by guest on April 9, 2018

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Figure 5. Srcasm promotes transcription from serum-response elements. A) Human

primary keratinocytes were transiently transfected with plasmids constituting an Elk-1

activated, serum-response element dependent luciferase assay. Approximately 16 hours

after transfection, cells were infected with Ad-Srcasm or Ad-Con. After 18 hours of

infection, the cells were stimulated for 5 hrs with hEGF (100 ng/ml) and then lysed.

Firefly luciferase activity was determined and the values reported are the numbers

directly obtained from the luminometer using Simplicity 2.0 software. The numerical

luciferase values obtained for the various conditions are as follows: uninfected cells: 13.3

+/- 1.2; +EGF 19.7 +/- 10.0; Ad-Con: 127.3 +/-28.2; +EGF 205.3 +/- 36.4; Ad-Srcasm:

192.3 +/- 41.2; +EGF 367.7 +/- 24.8. Data representative of three experiments. *-p value

<0.05. B) Levels of Srcasm protein in lysates utilized for luciferase assay.


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Figure 6. Increased Srcasm expression decreases keratinocyte proliferation. A)

Subconfluent PHKs cultured in MCDB complete media were infected with Ad-Srcasm or

Ad-Con (MOI=200), recovered overnight, and then were labeled with 10uM BrdU for

3hrs. Cells were harvested and stained with FITC conjugated α-BrdU antibody and then

labeled with 7-AAD. Cells were analyzed by flow cytometry to determine S-phase

fraction; R4 gate represents cells in “S” phase. N=2, with triplicate samples. B)

Percentage of keratinocytes in the “S” phase is displayed as a bargraph. *-p value <0.05.

C) PHKs treated as in A were subjected to growth factor deprivation (24 hours) or EGF

stimulation (10 ng/ml) for the indicated times. Cells were labeled with propidium iodide

and subjected to flow cytometric analysis to determine S-phase fraction. N=2. *-p value

<0.05


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Figure 7. Srcasm promotes keratinocyte differentiation by decreasing BrdU staining

and increasing filaggrin expression. A) Primary human keratinocytes cultured in 2-

well chamber slides were labeled with 10uM BrdU for 3hrs and double-stained with the

following: left panel, α-BrdU (red) and α-Srcasm (green), middle panel, α-BrdU (red)

and α-filaggrin (green). Keratinocytes cultured on chamber slides were also stained with

α-Srcasm (red) and α-filaggrin (green), confocal image, right pane. DAPI is the nuclear

counterstain. B) Western blot analysis correlates increased levels of Srcasm with

increased levels of filaggrin. Keratinocytes were infected with Ad-Con or Ad-Srcasm at

MOI of 200. Cells were lysed at the indicated times post- infection. Lysates were

subjected to western blot analysis using α-filaggrin, α-Srcasm, and α-β actin.


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Figure 8. Srcasm protein levels decrease in human cutaneous neoplasia. A)

Specificity of the Srcasm antisera is demonstrated in western blotting of COS cell lysates

containing endogenous (-) or tranfected (+) Srcasm; preincubation of antisera with 1µM

Srcasm fusion protein blocks binding. B) Pre-immune IgG exhibits non-specific staining

of stratum corneum and very weak staining of the epidermis. C) Affinity purified α-

Srcasm stains normal (unremarkable, UNR) epidermis, but not dermis or stratum

corneum. D) Actinic keratosis (AK) demonstrates decreased staining compared to

adjacent normal epidermis (UNR). E) Squamous cell carcinoma in-situ (SCC-IS)

demonstrates decreased Srcasm staining compared to adjacent normal epidermis (UNR).

F) Squamous cell carcinoma (SCC) demonstrates decreased Srcasm staining compared to

adjacent normal epidermis (UNR). G) Western blotting demonstrates that normal skin

has increased Srcasm levels compared to SCC. Lysates derived from unremarkable skin

and SCC tissue were subjected to western blot analysis with α-Srcasm and α-β actin.


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Figure 9. Schematic model of Srcasm function. EGF binding to the EGFR promotes

SFK activation leading to Srcasm phosphorylation. Higher Srcasm leve ls enhance SFK

activation resulting in increased phosphorylation at Y416. Enhanced SFK activity and

Srcasm levels promote signaling through p44/42 MAP kinases and Elk-1. Persistent

signaling through this pathway in primary keratiocytes is associated with decreased DNA

synthesis and increased filaggrin expression.


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Dans and John SeykoraWeijie Li, Chris Marshall, Lijuan Mei, Leonard Dzubow, Chrysalene Schmults, Michael

Srcasm modulates EGF and Src-kinase signaling in keratinocytes

published online December 3, 2004J. Biol. Chem.

10.1074/jbc.M406546200Access the most updated version of this article at doi:

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srcasm modulates egf and src-kinase signaling in keratinocytes

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