Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein.     

Global and Site-Specific Effect of Phosphorylation on Protein Turnover

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Regulation of l-DOPA Biosynthesis by Site-Specific Phosphorylation of Tyrosine Hydroxylase in AtT-20 Cells Expressing Wild-Type and Serine 40-Substituted Enzyme

Abstract: De novo l -DOPA biosynthesis was studied in stably transfected AtT-20 cells expressing wild-type- or [Leu⁴⁰]-recombinant tyrosine hydroxylase (rTH). Basal rates of DOPA accumulation were much higher by cells expressing rTH in which Leu was substituted for Ser⁴⁰ (S40L-rTH) than by those expressing wild-type rTH (WT-rTH). Treatment of WT-rTH cells with forskolin produced an increase in DOPA accumulation and a concomitant increase in WT-rTH phospho-Ser⁴⁰ content, whereas DOPA production by cells expressing S40L-rTH was entirely unaffected by forskolin. After forskolin treatment of ³²P_i-prelabeled cells, WT-rTH was phosphorylated at Ser⁸, Ser¹⁹, Ser³¹, and Ser⁴⁰, whereas ³²P incorporation into S40L-rTH was restricted to Ser⁸, Ser¹⁹, and Ser³¹. Relatively prolonged treatment of AtT-20 cells expressing WT-rTH with either a depolarizing agent (elevated potassium) or a phosphatase inhibitor (okadaic acid) increased DOPA production and increased the phosphorylation state of Ser⁴⁰; but, unlike forskolin, these treatments also increased DOPA production by cells expressing S40L-rTH. Thus, the present studies demonstrate that Ser⁴⁰ phosphorylation mediates forskolin-induced increases in DOPA biosynthesis directly but that mechanisms other than Ser⁴⁰ phosphorylation can mediate the increases in DOPA biosynthesis produced either by depolarization or by protein phosphatase inhibition.     

Phosphorylation of Serine 402 Regulates RacGAP Protein Activity of FilGAP Protein

FilGAP is a Rho GTPase-activating protein (GAP) that specifically regulates Rac. FilGAP is phosphorylated by ROCK, and this phosphorylation stimulates its RacGAP activity. However, it is unclear how phosphorylation regulates cellular functions and localization of FilGAP. We found that non-phosphorylatable FilGAP (ST/A) mutant is predominantly localized to the cytoskeleton along actin filaments and partially co-localized with vinculin around cell periphery, whereas phosphomimetic FilGAP (ST/D) mutant is diffusely cytoplasmic. Moreover, phosphorylated FilGAP detected by Phos-tag is also mainly localized in the cytoplasm. Of the six potential phosphorylation sites in FilGAP tested, only mutation of serine 402 to alanine (S402A) resulted in decreased cell spreading on fibronectin. FilGAP phosphorylated at Ser-402 is localized to the cytoplasm but not at the cytoskeleton. Although Ser-402 is highly phosphorylated in serum-starved quiescent cells, dephosphorylation of Ser-402 is accompanied with the cell spreading on fibronectin. Treatment of the cells expressing wild-type FilGAP with calyculin A, a Ser/Thr phosphatase inhibitor, suppressed cell spreading on fibronectin, whereas cells transfected with FilGAP S402A mutant were not affected by calyculin A. Expression of constitutively activate Arf6 Q67L mutant stimulated membrane blebbing activity of both non-phosphorylatable (ST/A) and phosphomimetic (ST/D) FilGAP mutants. Conversely, depletion of endogenous Arf6 suppressed membrane blebbing induced by FilGAP (ST/A) and (ST/D) mutants. Our study suggests that Arf6 and phosphorylation of FilGAP may regulate FilGAP, and phosphorylation of Ser-402 may play a role in the regulation of cell spreading on fibronectin.     

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.     

Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb^−/−mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb^−/− mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.     

Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449

Vascular smooth muscle Kv1 delayed rectifier K⁺ channels (K_DR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with ⁴⁴⁹pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide ⁴⁴⁵KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro ³²P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing K_DR channel modulation by agonists via PKA activation.Voltage-gated K⁺ channels (Kv)⁴ composed of members of the Kv1 family (Kv1) are expressed in vascular smooth muscle (VSM) and other excitable cells, where they play an important role in controlling membrane potential, a key mechanism for regulation of contraction and arterial diameter (1). Steady-state Ca²⁺ influx through voltage-gated Ca²⁺ channels, resulting in elevated cytosolic Ca²⁺ concentration and contraction, requires sustained depolarization. In contrast, Kv1 channel activation induces hyperpolarization, reduces voltage-gated Ca²⁺ channel open probability, and promotes relaxation (1–3).Phosphorylation of ion channels by protein kinases is an important mechanism by which ion channel gating (opening and closing) and, thereby, membrane potential are modulated by cellular signaling pathways. VSM Kv1 channels containing Kv1.2-Kv1.5 (4–6) alone or with Kv1.6 (7) contribute to control of arterial diameter by vasoconstrictor and vasodilator stimuli (8–13). For example, hyperpolarization and relaxation induced by elevations in intracellular (cAMP) are suppressed by the block of Kv1 channels (14–18), and the activity of Kv1-containing VSM K_DR is enhanced by protein kinase A (PKA) (10–13, 19–22). However, the molecular basis of this regulation is unclear.Previous studies showed that gating of Kv1.2 channels expressed in Xenopus oocytes is stimulated by PKA (23), similar to VSM K_DR channels, which contain this pore-forming Kv1 subunit (7–13). There are six potential PKA phosphorylation sites within the cytoplasmic regions of Kv1.2 (see Fig. 1, A and B) based on the consensus motif Arg/Lys-Xaa-Xaa-Ser/Thr (24). An N-terminal residue, Thr-46, was proposed to be the site of PKA phosphorylation, as mutation to valine (T46V) prevented modulation by β-adrenoreceptor activation or exposure to the catalytic subunit of PKA (cPKA) (23). However, this conclusion has been questioned based on structural analysis of the N-terminal domain and the positive shift in voltage-dependent gating produced by the T46V mutation (25). Sites Ser-440, Ser-441, and Ser-449 near the C terminus were recently identified to be phosphorylated in Kv1.2 immunoprecipitates derived from brain as well as transfected human embryonic kidney (HEK 293) and COS-1 cells using a proteomic approach (26). Phosphorylation of these residues was suggested to be important for trafficking to the cell membrane (26), but the kinase(s) involved was not identified. cAMP/PKA-dependent signaling was reported to alter surface expression of Kv1.2, but phosphorylation at Ser-440 and Ser-449 was not required (27).Open in a separate windowFIGURE 1.Identification of potential PKA consensus phosphorylation sites in Kv1.2 and immunoprecipitation of Myc-tagged wild-type and mutant Kv1.2. A, schematic of Kv1.2 with candidate PKA phosphorylation sites indicated by asterisks, and Ser/Thr residues relevant to the study are labeled. B, amino acid sequence of Kv1.2 indicating transmembrane domains (boxes), cytoplasmic domains (italics) assessed by MIDAS for phosphorylated residues, candidate PKA consensus sites (bold), and potential phosphorylated Ser/Thr residues (underlined). C, immunoblot (IB) analysis of untagged Kv1.2-WT and Myc-tagged WT and mutant constructs using anti-Kv1.2. D, immunoblot identification of untagged Kv1.2-WT and Myc-tagged WT and mutant constructs by anti-Kv1.2 in anti-Myc immunoprecipitates (IP).In the present study we used a combination of biochemical, mutagenesis, and electrophysiological analyses to 1) determine whether Kv1.2 is a substrate for PKA, 2) identify the PKA phosphorylation site(s) involved, and 3) determine whether phosphorylation of the identified residue(s) mediates PKA-dependent changes in Kv1.2 current amplitude.     

Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation

Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111. To investigate the possibility that ERK1/2 activation can enhance OCT4A degradation, we analyzed endogenous ubiquitination in cells transfected with FLAG-OCT4A alone or with constitutively active MEK1 (MEK1^CA), and we observed that the extent of OCT4 ubiquitination was clearly increased when MEK1^CA was coexpressed and that this increase was more evident after MG132 treatment. These results suggest an increase in OCT4A ubiquitination downstream of MEK1 activation, and this could account for the protein loss observed after FGF2 treatment and MEK1^CA transfection. Understanding and controlling the mechanism by which stem cells balance self-renewal would substantially advance our knowledge of stem cells.     

Protein Kinase C Activation Has Distinct Effects on the Localization, Phosphorylation and Detergent Solubility of the Claudin Protein Family in Tight and Leaky Epithelial Cells

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell–cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.     

Phosphorylation of Merkel Cell Polyomavirus Large Tumor Antigen at Serine 816 by ATM Kinase Induces Apoptosis in Host Cells

Merkel cell carcinoma is a highly aggressive form of skin cancer. Merkel cell polyomavirus (MCV) infection and DNA integration into the host genome correlate with 80% of all Merkel cell carcinoma cases. Integration of the MCV genome frequently results in mutations in the large tumor antigen (LT), leading to expression of a truncated LT that retains pRB binding but with a deletion of the C-terminal domain. Studies from our laboratory and others have shown that the MCV LT C-terminal helicase domain contains growth-inhibiting properties. Additionally, we have shown that host DNA damage response factors are recruited to viral replication centers. In this study, we identified a novel MCV LT phosphorylation site at Ser-816 in the C-terminal domain. We demonstrate that activation of the ATM pathway stimulated MCV LT phosphorylation at Ser-816, whereas inhibition of ATM kinase activity prevented LT phosphorylation at this site. In vitro phosphorylation experiments confirmed that ATM kinase is responsible for phosphorylating MCV LT at Ser-816. Finally, we show that ATM kinase-mediated MCV LT Ser-816 phosphorylation may contribute to the anti-tumorigenic properties of the MCV LT C-terminal domain.     

Ras-Regulated Hypophosphorylation of the Retinoblastoma Protein Mediates Neuronal Differentiation in PC12 Cells

Abstract: To investigate the role of the retinoblastoma protein pRB in neuronal differentiation, we have measured the accumulation of hypophosphorylated pRB in PC12 cells stimulated by nerve growth factor (NGF). NGF induced the accumulation of hypophosphorylated pRB within 30 min and the level peaked after 12 h. Viral Kiras, cyclic AMP (cAMP), and 12- O -tetradecanoylphorbol 13-acetate (TPA) also induced the hypophosphorylation of pRB, but epidermal growth factor and interleukin-6 did not. The extent of hypophosphorylation of pRB correlated well with the capacity of these factors to stimulate neurite outgrowth. The constitutively activated Ras induced persistent shift of the phosphorylation state of pRB toward hypophosphorylation. A dominant negative form of cHa-Ras suppressed significantly induction of the hypophosphorylation of pRB by NGF, but not by cAMP. Taken together, these results suggest that the hypophosphorylation of pRB triggered by NGF is mediated by a Ras-dependent pathway. Furthermore, microinjection of a monoclonal antibody specific for the hypophosphorylated form of pRB blocked the neurite outgrowth initiated by NGF. These results suggest a crucial role of pRB in withdrawal of cells from the cell cycle and in neuronal differentiation of PC12 cells.     

Serine Phosphorylation Sites on IRS2 Activated by Angiotensin II and Protein Kinase C To Induce Selective Insulin Resistance in Endothelial Cells

Protein kinase C (PKC) activation, induced by hyperglycemia and angiotensin II (AngII), inhibited insulin-induced phosphorylation of Akt/endothelial nitric oxide (eNOS) by decreasing tyrosine phosphorylation of IRS2 (p-Tyr-IRS2) in endothelial cells. PKC activation by phorbol ester (phorbol myristate acetate [PMA]) reduced insulin-induced p-Tyr-IRS2 by 46% ± 13% and, similarly, phosphorylation of Akt/eNOS. Site-specific mutational analysis showed that PMA increased serine phosphorylation at three sites on IRS2 (positions 303, 343, and 675), which affected insulin-induced tyrosine phosphorylation of IRS2 at positions 653, 671, and 911 (p-Tyr-IRS2) and p-Akt/eNOS. Specific PKCβ2 activation decreased p-Tyr-IRS2 and increased the phosphorylation of two serines (Ser303 and Ser675) on IRS2 that were confirmed in cells overexpressing single point mutants of IRS2 (S303A or S675A) containing a PKCβ2-dominant negative or selective PKCβ inhibitor. AngII induced phosphorylation only on Ser303 of IRS2 and inhibited insulin-induced p-Tyr911 of IRS2 and p-Akt/eNOS, which were blocked by an antagonist of AngII receptor I, losartan, or overexpression of single mutant S303A of IRS2. Increases in p-Ser303 and p-Ser675 and decreases in p-Tyr911 of IRS2 were observed in vessels of insulin-resistant Zucker fatty rats versus lean rats. Thus, AngII or PKCβ activation can phosphorylate Ser303 and Ser675 in IRS2 to inhibit insulin-induced p-Tyr911 and its anti-atherogenic actions (p-Akt/eNOS) in endothelial cells.     

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.     

Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.     

Tyrosine and Serine Phosphorylation of Dystrophin and the 58-kDa Protein in the Postsynaptic Membrane of Torpedo Electric Organ

Abstract: Dystrophin associates with a 58-kDa and an 87-kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87-kDa protein is a major phosphotyrosine-containing protein in these membranes. Immunoprecipitation of the 87-kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphoproteins. These phosphoproteins are identified as dystrophin and the 58-kDa protein. Monoclonal antibodies to dystrophin and the 58-kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58-kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58-kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58-kDa and 87-kDa proteins, may be modulated by tyrosine and serine protein Phosphorylation.