Preferred Site Of Phosphorylation Research Articles

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

Investigations on the effects of thermal and non-thermal treatments on ovalbumin conformation and allergenicity

The specific phosphorylation sites and degree of phosphorylation (DP) at each site are directly related to protein’s structure and functional properties. Thus, characterizing the introduced phosphate groups is of great importance. This study was to monitor the phosphorylation sites, DP and the number of phosphorylation sites in P-Oval achieved by dry heating in the presence of pyrophosphate for 1, 2 and 5 days by using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Two phosphorylation sites were found in natural ovalbumin, but the number of phosphorylation sites increased to 8, 8 and 10 after dry-heating phosphorylation for 1, 2 and 5 days, respectively. In addition, dual-phosphorylated peptides were detected for samples without extensive heating. The phosphorylation sites were found to be mainly on Ser residues, which could be the preferred phosphorylation site for dry heating in the presence of pyrophosphate.

Identification and quantification of the phosphorylated ovalbumin by high resolution mass spectrometry under dry-heating treatment

The specific phosphorylation sites and degree of phosphorylation (DP) at each site are directly related to protein’s structure and functional properties. Thus, characterizing the introduced phosphate groups is of great importance. This study was to monitor the phosphorylation sites, DP and the number of phosphorylation sites in P-Oval achieved by dry heating in the presence of pyrophosphate for 1, 2 and 5days by using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Two phosphorylation sites were found in natural ovalbumin, but the number of phosphorylation sites increased to 8, 8 and 10 after dry-heating phosphorylation for 1, 2 and 5days, respectively. In addition, dual-phosphorylated peptides were detected for samples without extensive heating. The phosphorylation sites were found to be mainly on Ser residues, which could be the preferred phosphorylation site for dry heating in the presence of pyrophosphate.

Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures.

This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K + current ( I Kr) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 μM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 μM) and the Src-family kinase inhibitor PP2 (10 μM) remarkably inhibited hERG channel current ( I hERG), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I hERG by PP2 and/or AG556. Our results demonstrate the novel information that I hERG is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.

Abnormal expression of Rb pathway–related proteins in salivary gland acinic cell carcinoma

Salivary gland acinic cell carcinoma (ACC) is a relatively rare neoplasm, and limited information is available regarding its molecular pathogenesis. Because the deregulation of Rb pathway is common to most human tumors, we immunohistochemically investigated the expression of Rb pathway–related proteins, including Rb, Rb proteins phosphorylated at serine 780 and 795 (pRb-S780 and pRb-S795, respectively), cyclin D1, and p16 INK4a in 18 cases of ACC. The expression of topoisomerase II– α and Ki-67 was also examined to evaluate cell proliferation. All the ACCs exhibited substantial numbers of positive cells against Rb antibody that recognizes both unphosphorylated and phosphorylated Rb proteins. The numbers of positive cells for pRb-S795 and cyclin D1 significantly increased in ACCs as compared with normal salivary glands. Double immunofluorescent staining demonstrated that pRb-S795 was colocalized with cyclin D1 in most tumor cells. However, neither significant change of the expression of Rb protein phosphorylated at serine 780 nor its colocalization with cyclin D1 was observed. The loss of p16 INK4a is infrequent, but its expression was correlated with phosphorylated Rb proteins. Our results suggest that serine 795 but not serine 780 is the preferred phosphorylation site induced by cyclin D1. This phosphorylation appeared to be critical for inactivation of Rb-mediated growth suppression and may play an important role in the pathogenesis of ACC.

Modulation of the Cardiac Sodium Channel Na V 1.5 by Fyn, a Src Family Tyrosine Kinase

Dynamic modulation of ion channels can produce dramatic alterations of electrical excitability in cardiac myocytes. This study addresses the effects of the Src family tyrosine kinase Fyn on Na(V)1.5 cardiac sodium channels. Sodium currents were acquired by whole cell recording on HEK-293 cells transiently expressing Na(V)1.5. Acute treatment of cells with insulin caused a depolarizing shift in steady-state inactivation, an effect eliminated by the Src-specific tyrosine kinase inhibitor PP2. Sodium channels were coexpressed with either constitutively active (Fyn(CA)) or catalytically inactive (Fyn(KD)) variants of Fyn. Fyn(CA) caused a 10-mV depolarizing shift of steady-state inactivation compared with Fyn(KD) without altering the activation conductance-voltage relationship. Comparable effects of these Fyn variants were obtained with whole-cell and perforated-patch recording. Tyrosine phosphorylation of immunoprecipitated Na(V)1.5 was increased in cells expressing Fyn(CA) compared with Fyn(KD). We show that Fyn is present in rat cardiac myocytes, and that Na(V)1.5 channels from these myocytes are tyrosine-phosphorylated. In HEK-293 cells the effect of Fyn(CA) on Na(V)1.5 inactivation is abolished by the single point mutation Y1495F, a residue located within the cytoplasmic linker between the third and fourth homologous domains of the sodium channel. We provide evidence that this linker is a substrate for Fyn in vitro, and that Y1495 is a preferred phosphorylation site. These results suggest that cardiac sodium channels are physiologically relevant targets of Src family tyrosine kinases.

Sequential phosphorylation of visual arrestin in intactLimulusphotoreceptors: Identification of a highly light-regulated site

The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. We found that light increased the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. We speculate that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane.

Vasodilator-stimulated phosphoprotein regulates proliferation and growth inhibition by nitric oxide in vascular smooth muscle cells.

Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Mechanism of Janus kinase 3-catalyzed phosphorylation of a Janus kinase 1 activation loop peptide

A high-affinity IL-2 receptor requires two Janus protein tyrosine kinases (JAKs) for IL-2 signal transduction: JAK1 and JAK3. Since transphosphorylation of the two kinases is presumed to occur after receptor engagement we examined the phosphorylation by recombinant JAK3 of a peptide substrate corresponding to the JAK1 activation loop (KAIETDKEYYTVKD), which has two adjacent tyrosines. Mass spectral analysis of the enzymatically phosphorylated peptide showed that the second tyrosine was phosphorylated at a 30-fold greater rate than the first tyrosine. Moreover, no doubly phosphorylated peptide was detected by this analysis. Kinetic analysis of the reactions of singly phosphorylated JAK1 activation loop peptides showed that phosphorylating the first or second tyrosine decreased the k cat/ K m for the phosphorylation of the other 115- and 26-fold, respectively. Singly changing each side chain of the KEYYTV portion of the peptide to a methyl group (alanine) yielded substrates comparable to the wild-type sequences in all cases except that of the first or second tyrosine, which showed a 153- or 70-fold drop in k cat/ K m, respectively. Using libraries of immobilized peptides with all 20 naturally occurring amino acids substituted for Y9 or T11 showed that the JAK3 tolerated substitution at T11 but prefers large hydrophobic amino acids at Y9. These results show that JAK3 does not act processively on the JAK1 activation loop in vitro and illustrate the role of Y9 in the recognition of the preferred site of phosphorylation which is Y10.

Pim-1 associates with protein complexes necessary for mitosis.

The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the 'kinase-dead' mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.

Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2

Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7 S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with [gamma-(32)P]ATP. This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification. The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA. The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II). In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2. Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA. There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation. TFIIIA immunoprecipitated from oocytes, which were incubated with [(32)P]orthophosphate, is also phosphorylated exclusively on serine residues. Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo. A recognition sequence for CK2, located at Ser(16) in the beta-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide. Assays in vitro with site-specific mutants of TFIIIA established that Ser(16) is the preferred site of phosphorylation, with some secondary modification at Ser(314).

Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2

Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with [γ-32P]ATP. This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification. The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA. The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II). In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2. Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA. There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation. TFIIIA immunoprecipitated from oocytes, which were incubated with [32P]orthophosphate, is also phosphorylated exclusively on serine residues. Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo. A recognition sequence for CK2, located at Ser16 in the β-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide. Assays in vitro with site-specific mutants of TFIIIA established that Ser16 is the preferred site of phosphorylation, with some secondary modification at Ser314.

Heterogeneity in the Phosphorylation of Human Death Receptors by p42mapk/erk2

Phosphorylation of murine CD120a by p42mapk/erk2 has been shown to inhibit its ability to initiate apoptosis while preserving signaling events such as NF-κB activation. Therefore, we sought to determine if p42mapk/erk2 was also capable of phosphorylating additional human death receptors within the TNF receptor superfamily. These studies showed that CD120a and DR3 are significantly phosphorylated by p42mapk/erk2 but Fas, DR4 and DR5 are not. Additionally, we demonstrated that (i) the p42mapk/erk2-dependent phosphorylation of CD120a and DR3 occurred on Ser and Thr residues, (ii) p42mapk/erk2 phosphorylated residues located in the membrane proximal regions but not the death domains of CD120a and DR3, (iii) Ser 253 is a preferred site of phosphorylation on CD120a, and (iv) the p42mapk/erk2-dependent phosphorylation of the DR3 cytoplasmic domain occurred exclusively at non-p42/44mapk/erk2/1 consensus sites. These findings suggest that human death receptors segregate into two groups along lines of phylogeny with respect to Ser/Thr phosphorylation by p42mapk/erk2.

Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1.

In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Cds1 (Chk2) tumour-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo and can phosphorylate Cds1 in vitro, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.

The Vasodilator-Stimulated Phosphoprotein (VASP): Target of YC-1 and Nitric Oxide Effects in Human and Rat Platelets

The effects of the different types of soluble guanylate cyclase (sGC) stimulators on the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) in both human and rat platelets were studied under in vitro and in vivo conditions. sGC-dependent VASP phosphorylation (at Ser(239) and Ser(157)) both by the new direct sGC stimulator YC-1 and by NO donors was examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS/PAGE) with different antibodies. One antibody, which recognizes VASP independent of its phosphorylation state, was used to detect the mobility shift of VASP caused by Ser(157) phosphorylation. The other antibody was specifically directed against VASP phosphorylated at Ser(239), the cGMP-dependent protein kinase (PKG) preferred phosphorylation site of VASP. In vitro YC-1 increased both VASP phosphorylation and cyclic guanosine monophosphate (cGMP) levels as did the NO donors 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO) and sodium nitroprusside (SNP). The combination of both types induced a synergistic effect in both VASP phosphorylation and cGMP increase. In rat platelets, similar effects could be shown in vitro. In vivo we observed a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat-tail bleeding time. Direct stimulators of sGC like YC-1 are on the one hand direct potent stimulators of the cGMP/PKG/VASP pathway in platelets and on the other hand synergize with NO, the physiologic stimulator of sGC. Therefore YC-1-like substances are interesting tools for the development of new cardiovascular drugs with vasodilatory and antithrombotic properties.

Defining the substrate specificity of cdk4 kinase-cyclin D1 complex.

cdk4 kinase-cyclin D1 complex (cdk4/D1) does not phosphorylate all of the sites within retinoblastoma protein (Rb) equally. Comparison of five phosphorylation sites within the 15 kDa C domain of Rb indicates that Ser795 is the preferred site of phosphorylation by cdk4/D1. A series of experiments has been performed to determine the properties of this site that direct preferential phosphorylation. For cdk4/D1, the preferred amino acid at the third position C-terminal to the phosphorylated serine/threonine is arginine. Substitution of other amino acids, including a conservative change to lysine, has dramatic effects on the rates of phosphorylation. This information has been used to mutate less favorable sites in Rb, converting them to sites that are now preferentially phosphorylated by cdk4/D1. A conserved site at Ser842 in the related pocket protein p107 is also preferentially phosphorylated by cdk4/D1. Although Rb and p107 differ significantly in sequence, the Rb Ser795 site can replace the p107 Ser842 site without affecting the rate of phosphorylation. These results suggest that although a determinant of specificity resides in the sequences surrounding the phosphorylated site, the structural context of the site is also a critical parameter of specificity.

Defining the minimal portion of the retinoblastoma protein that serves as an efficient substrate for cdk4 kinase/cyclin D1 complex.

We have determined the minimal portion of the retinoblastoma protein (Rb) that can serve as an efficient substrate for in vitro phosphorylation by cdk4 kinase-D1 cyclin. Kinetic measurements indicate that in vitro, a 15-kDa fragment that represents the C-terminus of Rb can serve equally well as a substrate when compared with the larger 56-kDa fragment of Rb, which contains the A, B and C domains. By comparison, peptide substrates appear to be 1000-fold less efficient. Furthermore, mutational analysis indicates that not all of the five phosphorylation sites within this minimal C domain are phosphorylated equally by cdk4/D1. Ser795 is the preferred phosphorylation site, whereas the four remaining sites Ser807, Ser811, Thr821 and Thr826 are phosphorylated to a much lesser degree. Truncations of the C domain from the carboxy terminus indicate that almost all of this domain is required for efficient phosphorylation. These data suggest that the structural context of the phosphorylation site within the substrate is critical for its phosphorylation by the cdk4/D1 kinase.

SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells.

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

Two distinct actin-binding sites of smooth muscle calponin.

Amino acid residues 145-163 of calponin have been proposed as a putative actin-binding site [Mezgueldi, M., Mendre, C., Calas, B., Kassab, R. & Fattoum, A. (1995) J. Biol. Chem. 270, 8867-8876]. Our previous work demonstrated that a fragment of calponin, which corresponded to the first repeated region of calponin and contained the preferred site of phosphorylation by protein kinase C [Nakamura, F., Mino, T., Yamamoto, J., Naka, M. & Tanaka, T. (1993) J. Biol. Chem. 268, 6194-6201] enhanced the Ca2+-induced contraction of permeabilized smooth muscle [Itoh, T., Suzuki, A., Watanabe, Y., Mino, T., Naka, M. & Tanaka, T. (1995) J. Biol. Chem. 270, 20400-20403]. In the present study, we compared the interactions with actin of a synthetic peptide (Lys172-His187) that encompassed the first repeated region with those of three other synthetic peptides. Lys172-His187 inhibited the binding of calponin to F-actin in a concentration-dependent manner but not the binding of caldesmon. Gly141-Gly160, including the above-mentioned putative actin-binding site, also competed with intact calponin to the same extent as Lys172-His187. Inhibition of actomyosin MgATPase activity was observed only with Gly141-Gly160. Lys172-His187 and other tested peptides had no effect. However, Gly141-Gly160 and Lys172-His187 reduced the fluorescence intensity of pyrene-labeled F-actin with approximately equal potency. Moreover, Lys172-His187 was able to reverse the inhibition of actomyosin MgATPase activity by calponin. Lys172-His187 was phosphorylated stoichiometrically by protein kinase C and phosphorylation of this peptide decreased its actin-binding activity. These observations suggest the direct involvement of two distinct actin-binding sites, with different regulatory functions, in the interactions of calponin with actin.