LMP2A Induces Expression and Stability of p63α in Epithelial Cells
Several signaling motifs have been identified in LMP2A including a potential src kinase binding site (YEEA), an immunoreceptor tyrosine-based activation motif (ITAM) that binds the Syk tyrosine kinase, and two PY motifs that bind WW domain-containing ubiquitin ligases. LMP2A has previously been shown to inhibit differentiation in epithelial cells and the PY motif is required for the inhibition of involucrin expression upon differentiation in human foreskin keratinocytes (Morrison & Raab-Traub, 2005
; Scholle et al., 2000
). The ubiquitin ligase Itch interacts with the LMP2A PY motifs and regulates degradation of LMP2A and LMP2A-associated signaling mediators (Ikeda et al., 2003
; Ikeda et al., 2000
; Longnecker et al., 2000
; Rossi et al., 2006a
; Rossi et al., 2006b
). One target of Itch is p63, a transcription factor belonging to the p53 family, that has been identified as a key regulator of epithelial cell growth and differentiation (Candi et al., 2008
; Candi et al., 2007
; Medawar et al., 2008
; Murray-Zmijewski et al., 2006
; Ogawa et al., 2008
; Truong & Khavari, 2007
). ΔNp63α is associated with impaired epithelial cell differentation and with nasopharyngeal carcinoma, and as such may provide a key target for LMP2A signaling. To determine whether LMP2A affects ΔNp63α, its expression and stability were studied in HaCaT cells stably expressing LMP2A.
Using antibody specific for total p63α, immunoblotting of whole cell lysates revealed that LMP2A increased the protein levels of an approximately 72 kd protein, compared to pBabe, consistent with the size of ΔNp63α (). Antibody specific for ΔNp63 indicated that the 72 kd protein was ΔNp63α and quantitation relative to actin indicated that LMP2 increased the levels approximately 50% (). The LMP2A-induced increase in ΔNp63α expression was impaired by mutation of the LMP2A PY motifs or by deletion of the ITAM motif (). In contrast, the YEAA motif did not impair the increased ΔNp63α expression. The ITAM mutant that also lacks one PY domain was consistently expressed at higher levels perhaps reflecting its impaired activity and interaction with ubiquitin ligases. These findings suggest that the LMP2A-induced increase in ΔNp63α levels required PY and ITAM. Quantitative RT-PCR that distinguished the two distinct amino terminal forms revealed that the ΔNp63 isoform was predominant in HaCaT cells with essentially no expression of TAp63 (), further implicating ΔNp63α as the isoform targeted by LMP2A. Importantly, LMP2A did not increase the relative transcription of either form (data not shown), suggesting that ΔNp63α regulation by LMP2A was post-transcriptional.
LMP2A Increases Expression and Stability of p63α
To determine the effect of LMP2A on p63α protein stability, HaCaT cells expressing pBabe or LMP2A were treated with cycloheximide to inhibit protein synthesis, and p63α expression was determined by western blot. pBabe cells had a time-dependent decrease in p63α expression while in LMP2A cells, p63α expression did not decrease in the presence of a protein synthesis inhibitor, suggesting LMP2A increased the expression p63α through effects on its stability (). A half-life for p63α in LMP2A cells could not be calculated because a slight increase in p63α expression relative to actin from time 0 to 24 hours gave a line of best fit with a positive slope, indicating no degradation of p63α (). LMP2A expression decreased in a time-dependent manner following cycloheximide treatment indicating that LMP2A did not impair the protein degradation machinery in HaCaT cells (). The increased stability of ΔNp63α has also been described in NPC cell lines (Guo et al., 2006
To determine if LMP2A-induced p63α protein stability was caused by impaired proteasome-mediated degradation, pBabe and LMP2A expressing cells were treated with the proteasome inhibitor MG132. In pBabe cells, treatment with MG132 increased p63α expression compared to treatment with the DMSO vehicle control to levels equivalent to those in LMP2A expressing cells indicating that p63α is regulated by proteasome-mediated degradation (). In contrast, p63α levels in LMP2A cells were unchanged when treated with MG132 compared with DMSO and were similar to p63α levels in pBabe cells treated with MG132 (). These findings suggest that LMP2A impairs proteasome-mediated degradation of p63α in pBabe cells (). The Itch ubiquitin ligase that is known to regulate p63 was increased by LMP2A and inhibition of the proteasome did not affect Itch levels ().
LMP2A and p63α Interact in Cytoplasmic and Nuclear Membrane Fractions in Epithelial Cells
To assess the effects of LMP2A on the subcellular localization of Itch and p63α, HaCaT cells stably expressing LMP2A, LMP2A mutants, and ΔNp63α were fractionated into cytoplasm, nucleoplasm, and nuclear membrane. Protein expression was determined by western blot () and the levels of the specific proteins in each fraction are presented as % of the total individual protein with standard error calculated from 3 or more experiments (). The purity of the fractions was determined using the endoplasmic reticulum marker GRP78 and the nuclear membrane marker emerin (data not shown). In the pBabe vector control cell, Itch was predominantly detected in the cytoplasm and nucleoplasm fractions with low expression detected in the nuclear membrane (). Stable expression of LMP2A, PY, YEEA, or ΔNp63α did not affect Itch localization or levels, however loss of the ITAM motif decreased cytoplasmic and increased nuclear expression of Itch ().
LMP2A Immunoprecipitates with p63α in Cytoplasmic and Nuclear Membrane Fractions
In the vector control cells, p63α localized primarily to the cytoplasm and nuclear membrane fractions (). Stable expression of LMP2A, PY, ITAM, YEEA, or ΔNp63α did not significantly affect p63 localization (). However, the low levels of expression of p63α in pBabe, ITAM, and ΔNp63α cells compared with LMP2A-induced expression required longer exposures to detect bands for quantitation (). The lower overall expression of p63α in pBabe and ITAM cells was consistent with the lower expression detected in whole cell lysates (). Full length LMP2A and all LMP2A mutants were predominately cytoplasmic with some expression in the nuclear membrane fraction ().
To determine the potential interaction of LMP2A and p63α, LMP2A was immunoprecipitated using cytoplasm from the pBabe control cells and from the cytoplasm, nucleoplasm, and nuclear membranes from LMP2A expressing HaCaT cells. Itch and p63α were detected with LMP2A precipitated from the cytoplasm and nuclear membrane (). Neither Itch nor p63α were detected in the immunoprecipitated material from the pBabe lysates with the LMP2 antibody or by using beads alone in the absence of antibody indicating that their precipitation required LMP2 and did not reflect a non-specific interaction of Itch or p63α with the LMP2 antibody. These findings indicate that LMP2A interacts with endogenous p63 and confirm the interaction of LMP2A and Itch that has been previously described (Ikeda et al., 2003
; Ikeda et al., 2000
; Longnecker et al., 2000
The ability of LMP2A to interact with the ΔNp63α isoform was evaluated in HEK293 cells, that do not endogenously express detectable levels of p63α (data not shown), transfected with an expression construct for ΔNp63α. Endogenous Itch was detected in the cytoplasm, nucleoplasm, and nuclear membrane of the vector control HEK293 cells (). Transiently expressed LMP2A was predominantly detected in the nuclear membrane compared to the stably expressing LMP2A HaCaT cells. Localization of transiently expressed ΔNp63α was also localized primarily to the nuclear membrane with equivalent levels of detection in the cytoplasm and nucleoplasm (). The different localization of ΔNp63α and LMP2A in transient and stable expression systems suggests that initial protein expression is detected at the nuclear membrane, whereas stably expressed or endogenous proteins associate with cytoplasmic components. Surprisingly, the relative levels of nuclear ΔNp63α decreased in the presence of LMP2A.
LMP2A Associates with p63α in Cytoplasm and Nuclear Membrane and Requires the PY and ITAM Signaling Motifs
The interaction of ΔNp63α with Itch and LMP2 was evaluated in the cytoplasm, nucleoplasm, and nuclear membrane fractions of the transiently transfected HEK293 cells. Itch interacted withΔNp63α in the nuclear membrane where ΔNp63α was predominantly expressed (). Similarly, LMP2A also interacted with ΔNp63α at the nuclear membrane. While protein localization differed in transient and stable expression systems, LMP2A and ΔNp63α interacted under both conditions.
To determine which signaling motifs were required for the interaction of LMP2A with p63α, LMP2A was immunoprecipitated from HEK293 cells transiently expressing full-length or mutated LMP2A. ΔNp63α was detected at equivalent levels with immunoprecipitated LMP2A or LMP2A with the mutated YEEA motif but was significantly decreased with the PY and ITAM mutations (). Similarly, the interaction of Itch with LMP2A also required the PY and ITAM motifs (). The decreased immunoprecipitation of both ΔNp63α and Itch with these LMP2 mutants confirmed that Itch and ΔNp63α do not nonspecifically precipitate with the LMP2A antisera. The low level of Itch detected with LMP2A likely reflects that only a subset of the total Itch is present in the nuclear membrane where LMP2A is predominantly detected. The requirement of PY and ITAM for both interactions suggests that p63α, Itch, and LMP2A may exist as a complex.
LMP2A Co-Localizes with p63α
The potential interaction of LMP2A and p63α was also evaluated in HEK293 cells transiently expressing LMP2A and ΔNp63α, and in HaCaT cells stably expressing LMP2A, using immunofluorescent staining to identify colocalization. In HEK293 cells (, panels ii through iv), LMP2A and p63α colocalized with striking perinuclear staining confirming the cell fractionation and co-immunoprecipitation analyses (). Similarly, LMP2A and endogenous p63α primarily had perinuclear co-localization in HaCaT cells, suggesting they interact at the nuclear membrane, with some interaction evident in the cytoplasm ( panels vi-viii). The less intense staining of both LMP2A and p63α in HaCaT cells is likely a consequence of more uniform, stable, and less abundant expression of these proteins, whereas both proteins were transiently expressed in HEK293 cells and stained 48 hours following transfection. Background staining with FITC-anti-rat or Cy3-anti-mouse antibodies was not detected in either HEK293 cells or HaCaT cells ( panels ix–xii).
LMP2A Expression and Association with p63α is Localized to the Nuclear Membrane
LMP2A Impairs Epithelial Cell Differentiation Induced by Calcium
To determine the effects of LMP2A and ΔNp63α on epithelial cell differentiation, HaCaT cells were grown for 3 weeks in calcium-free DMEM to decrease differentiation. In the original calcium-containing growth media, HaCaT cells expressed high levels of the early differentiation marker keratin 10 and the intermediate differentiation marker involucrin (, day –5). Following treatment for 3 weeks without calcium, HaCaT cells with or without LMP2A acquired an undifferentiated morphology (data not shown; (Deyrieux & Wilson, 2007
)) with reduced levels of keratin 10 and involucrin (). The relative levels of keratin 10 and involucrin relative to GAPDH confirmed this decrease and indicated that LMP2A did not significantly affect the expression of these markers in de-differentiated cells ().
LMP2A is Associated with Inhibition of Differentiation Marker Expression
It is known that differentiation can be induced in proliferating keratinocytes by exposure to calcium. The effects on expression of keratin 10 and involucrin mRNA in response to the addition of 2.8mM CaCl2
to the growth media of the de-differentiated HaCaT cells were determined. HaCaT pBabe cells had a slight time dependent increase in calcium-induced mRNA expression of the early differentiation marker keratin 1 () and a clear induction of mRNA for the intermediate differentiation marker involucrin (). Whereas expression of the early marker keratin 1 peaked at 48 hours, expression of the intermediate marker involucrin continued to increase for 96 hours in pBabe cells. In contrast, the mRNA for keratin 1 and involucrin in HaCaT LMP2A cells decreased in response to calcium and remained low over the duration of the experiment (). Interestingly, LMP2A expressing cells had higher mRNA levels for both markers at time zero, in particular for the early marker keratin 1, although the difference in involucrin was not at the protein level (, day 21). Involucrin protein expression increased in response to calcium over time in the pBabe control cells, however this induction of involucrin was impaired in cells stably expressing LMP2A confirming the decreased expression of involucrin mRNA (). The impaired induction of the differentiation markers keratin 1 and involucrin in the presence of LMP2A confirms that LMP2 inhibits differentiation (). Similarly, involucrin was not induced in HaCaT cells that stably expressed ΔNp63α indicating that ΔNp63α expression can mimic the effect of LMP2A on calcium-induced differentiation. Calcium-induced involucrin expression in the PY mutant was similar to that observed in pBabe cells, confirming that the PY motif is required for the inhibition of differentiation by LMP2A () (Morrison and Raab-Traub, 2005
To determine whether LMP2A-induced ΔNp63α was associated with the state of differentiation of epithelial cells, p63α protein expression was determined by western blot in HaCaT cells, normalized to actin, prior to de-differentiation and following de-differentiation (). The p63α protein levels were slightly lower in LMP2A compared to pBabe cells prior to de-differentiation (), suggesting that LMP2A does not induce p63α or promote a de-differentiated phenotype when it is expressed in differentiated cells. However, LMP2A clearly induced ΔNp63α in the de-differentiated cells and both LMP2A and ΔNp63α inhibited expression of differentiation markers after exposure to calcium. The ability of LMP2A to induce expression of ΔNp63α was impaired with mutation of the PY or ITAM signaling motifs (). This data supports LMP2A-induced p63α as a mediator for inhibiting keratinocyte progress through the calcium-induced differentiation pathway.