Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.     

A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes

Insulin signaling through phosphatidylinositol 3-kinase (PI 3-kinase) activates the protein kinase Akt through phosphorylation of its threonine 308 and serine 473 residues by the PDK1 protein kinase and the Rictor-mammalian target of rapamycin complex (mTORC2), respectively. Remarkably, we show here that the Rictor protein is also present in cultured adipocytes in complexes containing Myo1c, a molecular motor that promotes cortical actin remodeling. Interestingly, the Rictor-Myo1c complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Furthermore, while RNA interference-directed silencing of Rictor results in the expected attenuation of Akt phosphorylation at serine 473, depletion of Myo1c is without effect. In contrast, loss of either Rictor or Myo1c inhibits phosphorylation of the actin filament regulatory protein paxillin at tyrosine 118. Furthermore, Myo1c-induced membrane ruffling of 3T3-L1 adipocytes is also compromised following Rictor knockdown. Interestingly, neither the mTORC2 inhibitor rapamycin nor the PI 3-kinase inhibitor wortmannin affects paxillin tyrosine 118 phosphorylation. Taken together, our findings suggest that the Rictor-Myo1c complex is distinct from mTORC2 and that Myo1c, in conjunction with Rictor, participates in cortical actin remodeling events.     

Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.     

Protein serine and threonine phosphorylation,hyperactivation and acrosome reaction in in vitro capacitated hamster spermatozoa

Monoclonal antibodies against phosphoserine and phosphothreonine were used in the present study to investigate the changes in serine and threonine phosphorylation respectectively during capacitation of hamster spermatozoa. Immunoblot analysis of hamster spermatozoa capacitated in TALP, a medium that supports capacitation, showed that a set of four proteins of molecular weight 56, 63, 66, and 100 kDa was phosphorylated both at the serine and threonine residues. In addition, five other proteins of molecular weight 32, 39, 45, 53, and 61 kDa were phosphorylated specifically at the threonine residues. Of these nine proteins, the 100 kDa protein showed a time dependent or capacitation-dependent decrease in intensity which coincided with the percentage acrosome-reacted spermatozoa. In contrast, the 49 and 63 kDa threonine phosphorylated proteins showed increased phosphorylation coinciding with capacitation. H8 (a serine and threonine kinase inhibitor) had a transient effect on the phosphorylation of these two phosphothreonine proteins but inhibited acrosome reaction substantially all through the treatment period. Okadaic acid (OA) (a serine and threonine protein phosphatase inhibitor) inhibited hyperactivation but had no effect on acrosome reaction. In fact, OA stimulated acrosome reaction. Finally the immunofluorescence studies indicated localization of the serine phosphorylated proteins in tail as well as in head of the capacitated hamster spermatozoa whereas the threonine phosphorylated proteins were localized mostly in the tail of the spermatozoa. The findings of the present study suggest that serine/threonine phosphorylation and the enzymes responsible for regulating the level of phosphorylation play an important role in capacitation and capacitation-associated events namely hyperactivation and acrosome reaction. However, further studies are needed in order to establish the exact role of these proteins in capacitation of spermatozoa.     

Serine phosphorylation regulates disabled-1 early isoform turnover independently of Reelin

The Reelin-Disabled 1 (Dab1) signaling pathway plays an important role in neuronal cell migration during brain development. Dab1, an intracellular adapter protein which is tyrosine phosphorylated upon Reelin stimulation, has been directly implicated in the transmission and termination of Reelin-mediated signaling. Two main forms of Dab1 have been identified in the developing chick retina, an early isoform (Dab1-E) expressed in progenitor cells and a late isoform (Dab1-L, a.k.a. Dab1) expressed in differentiated cells. Dab1-E is missing two Src family kinase (SFK) phosphorylation sites that are critical for Reelin-Dab1 signaling and is not tyrosine phosphorylated. We have recently demonstrated a role for Dab1-E in the maintenance of retinal progenitor cells. Here, we report that Dab1-E is phosphorylated at serine/threonine residues independent of Reelin. Cdk2, highly expressed in retinal progenitor cells, mediates Dab1-E phosphorylation at serine 475 which in turn promotes ubiquitination-triggered proteasome degradation of Dab1-E. Inhibition of protein phosphatase 1 and/or protein phosphatase 2A leads to increased Dab1-E instability. We propose that Dab1 turnover is regulated by both Reelin-independent serine/threonine phosphorylation and Reelin-dependent tyrosine phosphorylation.     

Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1

TAR DNA-binding protein-43 (TDP-43) proteinopathy has been linked to several neurodegenerative diseases, such as frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis. Phosphorylated and ubiquitinated TDP-43 C-terminal fragments have been found in cytoplasmic inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions and amyotrophic lateral sclerosis patients. However, the factors and pathways that regulate TDP-43 aggregation are still not clear. We found that the C-terminal 15 kDa fragment of TDP-43 is sufficient to induce aggregation but the aggregation phenotype is modified by additional sequences. Aggregation is accompanied by phosphorylation at serine residues 409/410. Mutation of 409/410 to phosphomimetic aspartic acid residues significantly reduces aggregation. Inhibition of either proteasome or autophagy dramatically increases TDP-43 aggregation. Furthermore, TDP-43 aggregates colocalize with markers of autophagy and the adaptor protein p62/SQSTM1. Over-expression of p62/SQSTM1 reduces TDP-43 aggregation in an autophagy and proteasome-dependent manner. These studies suggest that aggregation of TDP-43 C-terminal fragments is regulated by phosphorylation events and both the autophagy and proteasome-mediated degradation pathways.     

Identification of major ERK-related phosphorylation sites in Gab1

Gab1 (Grb2-associated binder1) belongs to a family of multifunctional docking proteins that play a central role in the integration of receptor tyrosine kinase (RTK) signaling, i.e., mediating cellular growth response, transformation, and apoptosis. In addition to RTK-specific tyrosine phosphorylation, these docking proteins also can be phosphorylated on serine/threonine residues affecting signal transduction. Since serine and threonine phosphorylation are capable of modulating the initial signal one major task to elucidate signal transduction via Gab1 is to determine the exact localization of distinct phosphorylation sites. To address this question in this report we examined extracellular signal-regulated kinases 1/2 (ERK) specific serine/threonine phosphorylation of the entire Gab1 engaged in insulin signaling in more detail in vitro. To elucidate the ERK1/2-specific phosphorylation pattern of Gab1, we used phosphopeptide mapping by two-dimensional HPLC analysis. Subsequently, phosphorylated serine/threonine residues were identified by sequencing the separated phosphopeptides using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation. Our results demonstrate that ERK1/2 phosphorylate Gab1 at six serine/threonine residues (T312, S381, S454, T476, S581, S597) in consensus motifs for MAP kinase phosphorylation. Serine residues S454, S581, S597, and threonine residue T476 represent nearly 80% of overall incorporated phosphate. These sites are located adjacent to src homology region-2 (SH2) binding motifs (YVPM-motif: Y447, Y472, Y619) specific for the phosphatidylinositol 3kinase (PI3K). The biological role of identified phosphorylation sites was proven by PI3K and Akt activity in intact cells. These data demonstrate that ERK1/2 modulate insulin action via Gab1 by targeting serine and threonine residues beside YXXM motifs. Accordingly, insulin signaling is blocked at the level of PI3K.     

Rad23 Interaction with the Proteasome Is Regulated by Phosphorylation of Its Ubiquitin-Like (UbL) Domain

Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.     

Inhibition of Pin1 reduces glutamate-induced perikaryal accumulation of phosphorylated neurofilament-H in neurons

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.