31 research outputs found
Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy
Abstract Cardiac physiology and hypertrophy are regulated
by the phosphorylation status of many proteins, which
is partly controlled by a poorly defined type 2A protein
phosphatase-alpha4 intracellular signalling axis. Quantitative
PCR analysis revealed that mRNA levels of the type
2A catalytic subunits were differentially expressed in H9c2
cardiomyocytes (PP2ACb[PP2ACa[PP4C[PP6C),
NRVM (PP2ACb[PP2ACa = PP4C = PP6C), and
adult rat ventricular myocytes (PP2ACa[
PP2ACb[PP6C[PP4C). Western analysis confirmed
that all type 2A catalytic subunits were expressed in H9c2
cardiomyocytes; however, PP4C protein was absent in
adult myocytes and only detectable following 26S proteasome
inhibition. Short-term knockdown of alpha4 protein
expression attenuated expression of all type 2A catalytic
subunits. Pressure overload-induced left ventricular (LV)
hypertrophy was associated with an increase in both
PP2AC and alpha4 protein expression. Although PP6C
expression was unchanged, expression of PP6C regulatory
subunits (1) Sit4-associated protein 1 (SAP1) and (2)
ankyrin repeat domain (ANKRD) 28 and 44 proteins was
elevated, whereas SAP2 expression was reduced in
hypertrophied LV tissue. Co-immunoprecipitation studies
demonstrated that the interaction between alpha4 and
PP2AC or PP6C subunits was either unchanged or reduced
in hypertrophied LV tissue, respectively. Phosphorylation
status of phospholemman (Ser63 and Ser68) was significantly
increased by knockdown of PP2ACa, PP2ACb, or
PP4C protein expression. DNA damage assessed by histone
H2A.X phosphorylation (cH2A.X) in hypertrophied tissue
remained unchanged. However, exposure of cardiomyocytes
to H2O2 increased levels of cH2A.X which was
unaffected by knockdown of PP6C expression, but was
abolished by the short-term knockdown of alpha4 expression.
This study illustrates the significance and altered
activity of the type 2A protein phosphatase-alpha4 complex
in healthy and hypertrophied myocardium
Essential Roles of the Tap42-Regulated Protein Phosphatase 2A (PP2A) Family in Wing Imaginal Disc Development of Drosophila melanogaster
Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development
Kindlin-1 and -2 Directly Bind the C-terminal Region of β Integrin Cytoplasmic Tails and Exert Integrin-specific Activation Effects*S⃞
Integrin activation, the rapid conversion of integrin adhesion receptors
from low to high affinity, occurs in response to intracellular signals that
act on the short cytoplasmic tails of integrin β subunits. Talin binding
to integrin β tails provides one key activation signal, but additional
factors are likely to cooperate with talin to regulate integrin activation.
The integrin β tail-binding proteins kindlin-2 and kindlin-3 were
recently identified as integrin co-activators. Here we report an analysis of
kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails
and describe the effect of kindlin expression on integrin activation. We
demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin
β tails in pulldown binding assays. Our mutational analysis shows that
the second conserved NXXY motif (Tyr795), a preceding
threonine-containing region (Thr788 and Thr789) of the
integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the
kindlin FERM domain (kindlin-1 Trp612 and kindlin-2
Trp615) are required for direct kindlin-integrin interactions.
Similar interactions were observed for integrin β3 tails. Using
fluorescence-activated cell sorting we further show that transient expression
of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of
endogenous α5β1 or stably expressed αIIbβ3 integrins.
This inhibition is not dependent on direct kindlin-integrin interactions
because mutant kindlins exhibiting impaired integrin binding activity
effectively inhibit integrin activation. Consistent with previous reports, we
find that when co-expressed with the talin head, kindlin-1 or -2 can activate
αIIbβ3. This effect is dependent on an intact integrin-binding site
in kindlin. Notably however, even when co-expressed with activating levels of
talin head, neither kindlin-1 or -2 can cooperate with talin to activate
β1 integrins; instead they strongly inhibit talin-mediated activation. We
suggest that kindlins are adaptor proteins that regulate integrin activation,
that kindlin expression levels determine their effects, and that kindlins may
exert integrin-specific effects